ES2421132T3 - A non-structural NS3 / 4A fusion gene optimized for hepatitis C virus codon - Google Patents

Abstract

A purified or isolated nucleic acid comprising at least 200 consecutive nucleotides of the NS3 / 4A gene of SEQ ID No. 35 or its complement, wherein said nucleic acid is capable of inducing an immune response against HCV NS3 / 4A.

Description

A non-structural NS3 / 4A fusion gene optimized for hepatitis C virus codon

Field of the Invention

Aspects of the present invention relate to the creation of a new hepatitis C virus (HCV) NS3 / 4A gene that has been codon optimized for expression in humans. Embodiments include the codon optimized NS3 / 4A nucleic acid compositions containing said nucleic acids as well as methods of manufacturing and using the aforementioned compositions including, among others, diagnostics or for preparing a medicament for the treatment and prevention of HCV infection

Background of the invention

Viruses are intracellular parasites that require the biochemical machinery of a host cell to replicate and spread. All viral particles contain some generic information that encodes viral structural proteins and enzymes. The generic material can be single-stranded or double-stranded DNA or RNA. (Virology, Fields ed., Third edition, Lippencott-Raven publishers, pp. 72-83 (1996)). The viral nucleic acid is surrounded by a protein coat called capsid. (Id.) In some viruses, the capsid is surrounded by an additional layer composed of a lipid membrane, called a cover. (Id. At 83-95).

The typical life cycle of the virus begins with the infection of a host cell through the binding of the viral particle to a cell surface receptor and the internalization of the viral capsid. (Id. In 103). Accordingly, a virus host fan is limited to cells that express a suitable cell surface receptor. Once internalized, the virus particle is dismantled and its nucleic acid is transcribed, translated or replicated. (Id.) At this point, the virus can undergo lytic replication, in which new viral particles are formed and released from the infected cell. (Id. In 105-11). The influenza virus is a typical example of a virus that undergoes lytic replication immediately after infection of a host cell. (Id. In 1369-85).

Alternatively, a virus can enter the latent phase, what is called lysogeny, in which the genome replicates but few or no viral proteins are actually expressed and viral particles are not formed. (Id. In 21929). Herpesviruses, such as the Epstein-Barr virus, are typical examples of viruses that establish latent infection in host cells. (Id. At 229-34). In the end, for the virus to spread, it must exit lysogeny and enter the lytic phase. Viral particles that are released during the lytic phase infect other cells of the same individual or can be transmitted to another individual where they establish a new infection.

Since the life cycle of the virus comprises an intracellular phase and an extracellular phase, both humoral and cellular immune defense systems are important in fighting viral infections. (Id. At 467-73). Antibodies directed against viral proteins can block the interaction of virus particles with their cellular receptor or, on the other hand, interfere with internalization or release processes. (Id. In 471). An antibody capable of interfering with the virus's life cycle is called a neutralizing antibody.

During intracellular replication viral proteins are produced, which are foreign to the host cell and some of these proteins are digested by cellular proteases after coupling to a molecule of the Major Histocompatibility Complex (MHC) presented on the surface of the infected cell. (Id. At 350-58). Therefore, the infected cell is recognized by T lymphocytes, macrophages or NK cells and is killed before the virus replicates and spreads to adjacent cells. ((Id. 468-70) In addition, the presence of viral nucleic acids, most notably in the form of double stranded RNA, triggers the infected cell to shut down its translation machinery and produce antiviral signaling molecules known as interferons. ( Id. In 37679).

However, viruses have developed several means of avoiding the host's immune defense system. By establishing latency (i.e., lysogeny), for example, the virus does not enter the lytic phase and avoids the humoral immune defense system. (Id. In 224). During the latency phase, few viral proteins are produced and infected cells have only a minimal capacity to present to the lymphocytes and macrophages that surround them signs that they are in their infected state. (Id. At 225-26). Additionally, some viral proteins, more especially those produced during latency, develop polypeptide sequences that cannot be efficiently presented to the cell-mediated immune defense system. (Levitskaya et al., Nature 375: 685-88 (1995)). Finally, some viruses can actively interfere with the immune response of the infected host, for example by preventing surface expression of MHC molecules (Fruh et al., J. Mol. Med. 75: 18-27 (1997) ), or altering interferon signaling (Fortunato et al., Trends Microbiol. 8: 111-19 (2000)).

Particularly elusive are hepatitis viruses, which are not classified as family but are grouped based on their ability to infect liver cells. Hepatitis C virus (HCV) belongs to the Flaviviridae family of viruses with single stranded RNA. Virology supra., P. 945-51). The HCV genome is approximately 9.6 kb in length and encodes at least ten polypeptides. (Kato, Microb. Comp. Genomics, 5: 129-151 (2000)). Genomic RNA is translated into a single polyprotein that is then cleaved by viral and cellular proteases to give the functional polypeptides. (Id.) Polyprotein is cleaved into three structural proteins (core protein E1 and E2), p7 of unknown function and six non-structural proteins (NS) (NS2, NS3, NS4A / B, NSSA / B). (Id.) NS3 encodes a serine protease that is responsible for the proteolytic events required for virus maturation (Kwong et al., Antiviral Res., 41: 67-84 (1999)) and NS4A acts as a cofactor for the NS3 protease . (Id.) NS3 also exhibits NTPase activity and possesses RNA helicase activity in vitro. (Kwong et al., Curr. Top. Microbiol. Immunol., 242: 171-96 (2000)).

HCV infection usually progresses from an acute to a chronic phase. (Virology, supra, pages 104147). Acute infection is characterized by high viral replication and high viral load in liver tissue and peripheral blood. (Id. In 1041-42.) Acute infection is eliminated by the patient's immune defense system in approximately 15% of infected individuals; in the other 85%, the virus establishes a chronic persistent infection. (Lawrence, Adv. Intern. Med., 45: 65-105 (2000)). During the chronic phase replication occurs in the liver and some viruses can be detected in peripheral blood. (Virology, supra, pages 1042).

The development of strategies to evade the host's immune defense system is essential for the establishment of a persistent infection. HCV, as a single-stranded DNA virus, exhibits a high rate of mutations in the replication and transcription of its genome. (Id. In 1046). Therefore, it has been observed that antibodies produced during the lytic phase hardly neutralize virus strains produced during chronic infection. (Id.) Although it appears that HCV does not interfere with the processing of the antigen and its presentation on MHC-I molecules, the NS5A viral protein may be involved in repression of interferon signaling through inhibition of protein kinase. PKR (Tan et al., Virology, 284: 1-12 (2001)).

The infected host generates a humoral and cellular immune response against the HCV virus but in most cases the response does not prevent the establishment of chronic disease. After the acute phase, the infected patient produces antiviral antibodies, including neutralizing antibodies against the E1 and E2 coat proteins. (Id. In 1045). This antibody response is prolonged during chronic infection. (Id.) In chronically infected patients, the liver is also infiltrated with CD8 + and CD4 + lymphocytes. (Id. In 1044-45). Additionally, infected patients produce interferons as an early response to viral infection. (Id. In 1045). It is likely that the energy of the initial immune response against infection determines whether the virus will be eliminated or without the infection will progress to a chronic phase. (Pape et al., J. Viral. Hepat., 6 Supp. 1: 36-40 (1999)). Despite the efforts of others, the need for efficient immunogens and medications for the prevention and treatment of HCV infection is manifest.

A new HCV isolate was discovered. A new NS3 / 4A fragment of the HCV genome of a patient infected with HCV (SEQ ID No. 1) was cloned and sequenced. This sequence was found to be only 93% homologous with the most closely related HCV sequence. This peptide (SEQ ID No. 2) or fragments thereof containing any number of consecutive amino acids between at least 3-50 amino acids of SEQ ID No. 2 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive amino acids), nucleic acids encoding these molecules, vectors having said nucleic acids and cells having said vectors, nucleic acids or peptides. It was found that NS3 / 4A nucleic acid, fragments thereof and corresponding peptides are immunogenic. Vaccine compositions and immunogenic preparations comprising, consisting of or consisting essentially of the HCV peptide of SEQ ID No. 2 or fragments thereof (eg, SEQ ID NO. 14 and 15) containing any number are also disclosed. of consecutive amino acids between at least 3-50 amino acids of SEQ ID NO: 2 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45

or 50 consecutive amino acids) or a nucleic acid encoding said peptide or fragment.

NS3 / 4A peptide mutants were also created and found to be immunogenic. Some mutants are truncated versions of the NS3 / 4A peptide (eg, SEQ ID. No. 12 and 13) and others lack a proteolytic cleavage site (eg, SEQ ID. No. 3-11). These peptides (e.g., SEQ ID NO: 3-13) and fragments thereof containing any number of consecutive amino acids between at least 3-50 amino acids (e.g., 3, 4, 6, 8, 10 , 12, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive amino acids) of any one of SEQ ID NO. 3-13 (e.g., SEQ ID NO: 15-26), nucleic acids that these molecules encode, vectors that have said nucleic acids and cells that have said vectors, nucleic acids or peptides are also disclosed, as well as a vaccine composition

or immunogenic preparation comprising, consisting of or consisting essentially of at least one HCV peptide of SEQ ID NOS 3-11 or fragments thereof containing any number of consecutive amino acids between at least 3-50 amino acids (eg. , 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive amino acids) of any one of SEQ ID NO: 3-11 (e.g., SEC ID No. 16-26) or a nucleic acid encoding said peptides

or fragments

WO0213855 (TRIPEP AB) describes and claims a composition comprising ribavirin and an HCV peptide of SEQ ID NO: 17 (eg, claim 2).

WO03031588 and WO2004046175 describe HCV nucleic acid sequences.

Summary of the invention

The invention, in its broadest sense, is as defined in the independent claims.

An embodiment of the invention includes a nucleic acid encoding NS3 / 4A comprising a sequence that was optimized by the most frequently used codons in humans. The nucleic acid sequence of the codon optimized NS3 / 4A nucleic acid sequence (coNS3 / 4A) is provided in SEQ ID No. 35, while the peptide encoded by said nucleic acid sequence is provided in SEQ ID NO. 36. This nucleic acid does not correspond to any known HCV sequence or genome. It was found that the nucleic acid encoding codon-optimized NS3 / 4A had a homology of only 79%, in the region of nucleotide positions 3417-5475, with HCV-1 and contained a total of 433 different nucleotides. The NS3 / 4A peptide encoded by the codon optimized nucleic acid sequence had a homology of only 98% with HCV-1 and contained a total of 15 different amino acids. It was found that codon optimized nucleic acid generated a higher level of expression of NS3 and was found to be more immunogenic with respect to both humoral and cellular responses than the native NS3 / 4A gene.

Transient transfections of human HepG2 cells showed that the coNS3 / 4A gene gave 11 times higher levels of NS3 compared to the wtNS3 / 4A gene when the CMV promoter was used. It was also shown that the presence of NS4A enhances expression by the Semliki forest virus (SFV). SFV constructs containing wild NS3 / 4A were manufactured by isolating the wtNS3 / 4A gene by polymerase chain reaction as a SpeI-BStB1 fragment, which was inserted into the SpeI-BStB1 site of pSFV10Enh containing an enhancer sequence of the translation of 34 amino acids in length of the capsid followed by the cleavage peptide FMDV2a. The packaging of the recombinant RNA into rSFV particles was performed using a two-collaborator RNA system. Both codon optimization and amplification of SFV replicase-mediated mRNA resulted in an improvement in immunogenicity that is evidenced by higher levels of NS3 specific antibodies. This improved immunogenicity and also resulted in faster stimulation of cytotoxic T lymphocytes (CTL). Since HCV is a non-cytolytic virus, the functionality of the stimulated CLT responses was evaluated by in vivo exposure to syngeneic tumor cells expressing NS3 / 4A. Previous stimulation of a tumor protective immunity required endogenous production of the immunogen and CD8 + CTLs, but was independent of CD4 + B and T lymphocytes. This model confirmed the faster in vivo activation of a tumor-specific immunity of NS3 / 4A by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3 / 4A gene using a gene gun six to 12 days after tumor injection significantly reduced tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3 / 4A. Therefore, either or both of these approaches are preferred in a genetic vaccine against HCV based on NS3 / 4A.

Accordingly, aspects of the present invention include compositions that comprise, consist of

or consist essentially of the nucleic acid sequence provided by the sequence of SEQ ID No. 35. Preferred embodiments include, for example, compositions comprising, consisting of or consisting essentially of any number of consecutive nucleotides between at least 50 nucleotides of SEQ ID NO. 35 or a complementary sequence thereof (e.g., 50-100, 100-200, 200-500, 500-1000, 1000-1500, 1500-2079 or 1500-2112 consecutive nucleotides with the condition as cited in claim 2). Preferred embodiments also include compositions comprising, consisting of, consisting essentially of any number of consecutive nucleotides between at least 50 nucleotides of SEQ ID No. 35 or a complementary sequence thereof (e.g., at least 50, 60, 70 , 80, 90 or 100 consecutive nucleotides of SEQ ID No. 35) with the condition as cited in claim 2.

Manufacturing and use procedures of the compositions described herein are also provided. In addition to the methods of manufacturing the nucleic acids of the embodiment, other embodiments include methods of manufacturing immunogens and / or vaccine compositions that can be used to treat or prevent HCV infection. Some procedures are practiced, for example, by mixing an adjuvant with a nucleic acid antigen (e.g., HCV nucleic acid) as described above, to formulate a single composition (e.g., a composition of vaccine). Preferred procedures involve mixing ribavirin with an HCV gene disclosed herein.

Preferred methods of using the compositions described herein involve its use to prepare a suitable medicament to provide an animal that needs an immune response to HCV a sufficient amount of one or more of the embodiments of nucleic acid described herein. An approach, for example, identifies an animal that needs an immune response to HCV (e.g., an animal at risk or already infected with HCV, such as a human being) and provides an amount to that animal NS3 / 4A nucleic acid of the invention that is effective to enhance or facilitate an immune response to the viral hepatitis antigen. Additional procedures are practiced by identifying an animal that needs a potent immune response to HCV and providing said animal with a composition comprising a nucleic acid of the invention. In some embodiments, the composition described above also contains an amount of ribavirin that provides an adjuvant effect.

In still more embodiments, for example, the nucleic acid of the invention is formulated for administration by a gene gun to a subject mammal in need of an immune response to HCV. In some embodiments, an amount of ribavirin is mixed with the immunogenic DNA prior to release with the gene gun. In other embodiments, the immunogenic DNA is formulated to be administered by gene gun shortly before

or after administration of ribavirin or near the same site of DNA inoculation.

Brief description of the figures

Figure 1 is a graph showing in H-2d mice the antibody titer against NS3 as a function of time after the first intramuscular immunization. The diamonds indicate the antibody titer in mice immunized with NS3 / 4A-pVAX and the squares indicate the titer of antibodies in mice immunized with NS3pVAX.

Figure 2 shows the in vivo protection conferred by an immunization with NS3 / 4A-pVAX1 gene gun (4 Ig)

or MSLF1-pVAX1 (4 Ig). Mice were immunized with the respective plasmids and 14 days later the mice were exposed to an SP2 / 0 cell line expressing NS3 / 4A (approximately 10 cells / mouse). The tumor size was then measured through the skin daily after 6 days from exposure and data were plotted.

Figure 3 shows the in vivo protection conferred by two immunizations with NS3 / 4A-pVAX1 (4 Ig) or MSLF1-pVAX1 (4 Ig) gene guns. Mice were immunized with the respective plasmid on week zero and week four and 14 days after the last immunization, mice were exposed to an SP2 / 0 cell line expressing NS3 / 4A (approximately 106 cells / mouse). The tumor size was then measured through the skin daily after 6 days from exposure and data were plotted.

Figure 4 shows the in vivo protection conferred by three immunizations with NS3 / 4A-pVAX1 (4Ig) or MSLF1-pVAX1 (4 Ig) gene guns. Mice were immunized with the respective plasmid on week zero, week four and week eight 14 days after the last immunization, mice were exposed to an SP2 / 0 cell line expressing NS3 / 4A (approximately 106 cells / mouse ). The tumor size was then measured through the skin daily after 6 days from exposure and data were plotted.

Figure 5A is a graph showing the percentage of CTL mediated lysis of SP2 / 0 target cells as a function of the effect or proportion of the target. As the control immunogen phosphate buffered saline (PBS) was used.

Figure 5B is a graph showing the percentage of CTL-mediated lysis of SP2 / 0 target cells as a function of the effect or proportion of the target. As an immunogen, plasmid NS3 / 4A-pVAX was used.

Figure 6A is a graph showing the response of previously untreated splenic T cells that were stimulated with peptide coated RMA-S cells. The previously untreated splenic T cells were obtained from C57 / BL6 mice.

Figure 6B is a graph showing the response of spleen T cells stimulated with RMA-S cells coated with peptide. Splenic T cells were obtained from C57 / BL6 mice to which a single dose of 4 Ig of MSLF1-pVAX1 had been administered.

Figure 6C is a graph showing the response of spleen T cells stimulated with RMA-S cells coated with peptide. Splenic T cells were obtained from C57 / BL6 mice that had been administered a single 4 Ig dose of NS3 / 4A-pVAX1.

Figure 6D is a graph showing the response of previously untreated splenic T cells that were stimulated with peptide coated RMA-S cells. The previously untreated splenic T cells were obtained from C57 / BL6 mice.

Figure 6E is a graph showing the response of spleen T cells stimulated with RMA-S cells coated with peptide. Splenic T cells were obtained from C57 / BL6 mice to which two doses of 4 Ig of MSLF1-pVAX1 had been administered.

Figure 6F is a graph showing the response of spleen T cells stimulated with RMA-S cells coated with peptide. Splenic T cells were obtained from C57 / BL6 mice to which two doses of 4 Ig of NS3 / 4A-pVAX1 had been administered.

Figure 6G is a graph showing the response of previously untreated splenic T cells that were stimulated with EL-4 cells expressing NS3 / 4A. The previously untreated splenic T cells were obtained from C57 / BL6 mice.

Figure 6H is a graph showing the response of splenic T cells that were stimulated with EL-4 cells expressing NS3 / 4A. Splenic T cells were obtained from C57 / BL6 mice to which a single dose of 4 Ig of MSLF1-pVAX1 had been administered.

Figure 6I is a graph showing the response of splenic T cells that were stimulated with EL-4 cells expressing NS3 / 4A. Splenic T cells were obtained from C57 / BL6 mice that had been administered a single 4 Ig dose of NS3 / 4A-pVAX1.

Figure 6J is a graph showing the response of previously untreated splenic T cells that were stimulated with EL-4 cells expressing NS3 / 4A. The previously untreated splenic T cells were obtained from C57 / BL6 mice.

Figure 6K is a graph showing the response of splenic T cells that were stimulated with EL-4 cells expressing NS3 / 4A. Splenic T cells were obtained from C57 / BL6 mice to which two doses of 4 Ig of MSLF1-pVAX1 had been administered.

Figure 6L is a graph showing the response of splenic T cells that were stimulated with EL-4 cells expressing NS3 / 4A. Splenic T cells were obtained from C57 / BL6 mice to which two doses of 4 Ig of NS3 / 4A-pVAX1 had been administered.

Figure 7 is a graph showing the humoral response at 10 and 100 Ig of the recombinant non-structural protein (NS3) of hepatitis C virus (HCV), determined by the mean final titers, when a single dose of co-administered 1 mg of ribavirin

Figure 8 is a graph showing the humoral response at 20 Ig of the recombinant hepatitis C virus (HCV) non-structural protein 3 (NS3), determined by the mean final titers, when a single dose of 0 was coadministered. 1, 10 or 10 mg of ribavirin.

Figure 9 is a graph showing the effects of a single dose of 1 mg of ribavirin on the proliferative responses of NS3-specific lymph nodes, determined by in vitro recall responses.

Figure 10 shows the average responses of NS3-specific antibodies stimulated by immunizations with 4 Ig gene wtNS3 / 4A-pVAX1 and coNS3 / 4A-pVAX1, or s.c. injection. of 107 particles of wtNS3 / 4A-SFV in groups of ten H-2d mice (a). Mice were immunized on weeks zero and four. Values are given as the mean final antibody titers (± SD.). Also shown (v) are the IgG subclass patterns of groups of five mice immunized twice with wtNS3 / 4A-pVAX1 administered i.m., coNS3 / 4A-pVAX1 administered i.m. or by means of a gene gun and wtNS3 / 4A-SFV administered s.c. Values are given as the mean final antibody titers (± SD.) A "**" sign indicates a statistical difference of p <0.01, a "*" sign indicates a difference of p <0.05 and NS ( not significant) indicates absence of statistical difference (Mann-Whitney). The proportion of the title obtained is also provided by dividing the average final titer of IgG2a antibodies against NS3 by the mean final titer of IgG1 antibodies against NS3. A high proportion (> 3) indicates a response of type Th1 and a low proportion (<0.3) indicates a response of type Th2, while values within a three-fold difference of 1 (0.3 to 3) indicate a mixed Th1 / Th2 response.

Figure 11 shows a flow cytometry quantification of the precursor frequency of NS3 / 4th specific CD8 + T cells using the H-2Db: Ig fusion protein loaded with the peptide. In a) the average% of NS3-specific CD8 + T lymphocytes from groups of five mice immunized twice with wtNS3-pVAX1, wtNS3 / 4A-pVAX1 or coNS3 / 4A-pVAX1 using the gene gun is shown. A "*" sign indicates a difference of p <0.05 and NS (not significant) indicates the absence of statistical difference (Mann-Whitney). The raw data of individual mice representative of the groups indicated above (e, f and h) as well as of individual mice immunized once with coNS3 / 4A-pVAX1 (b) or wtNS3 / 4A-SFV (c) are also shown. In (d) and (g), non-immunized control mice of the different experiments are provided. In (i) and (j), splenocytes were re-stimulated for five days with the NS3 peptides before analysis. A total of 150,000-200,000 data points were collected and the percentage of CD8 + cells stained for H-2Db: Ig is indicated in parentheses in each dot plot.

Figure 12 shows the previous stimulation of CTL detectable in vitro in H-2b mice by gene gun immunization of plasmids wtNS3-pVAX1, wtNS3 / 4 and coNS3 / 4A or injection s.c. of particles wtNS3 / 4A-SFV. Groups of five to 10 H-2b mice were immunized once (a) or twice (b). The percentage of specific lysis corresponds to the percentage of lysis obtained with the RMA-S cells coated with the NS3 peptide (upper panel in

(a) and (b) or EL-4 cells expressing NS3 / 4A (lower panel in a and b) minus the percentage of lysis obtained with non-transfected EL-4 cells. Values for the proportions between effector cells: target (E: D) of 60: 1, 20: 1 and 7: 1 have been provided. Each line indicates an individual mouse.

Figure 13 shows the specificity of immune responses that inhibit the tumor stimulated by gene gun immunization (panel (a)). Groups of ten C57BL / 6 mice were left untreated or given two monthly immunizations with 4 Ig of coNS3 / 4A-pVAX1. Two weeks after the last immunization, the mice were injected subcutaneously with the parental EL-4 cell line or 106 EL-4 cells expressing NS3 / 4A. Tumor sizes were measured through the skin at 6, 7, 10, 11, 12 and 14 days after tumor injection. In (b), the population of functional effector cells in vivo was determined in groups of 10 C57BL / 6 mice immunized twice with the plasmid coNS3 / 4A-pVAX1 using a gene gun. In two groups, CD4 + or CD8 + T lymphocytes were depleted by administration of monoclonal antibodies one week before and during exposure with the EL-4 cell line expressing NS3 / 4th. Tumor sizes were measured through the skin at 5, 6, 8, 11, 13, 14 and 15 days after tumor injection. Values have been provided as the mean tumor size ± standard error. A "**" sign indicates a statistical difference of p <0.01, a "*" sign indicates a difference of p <0.05 and NS (not significant) indicates absence of statistical difference (the values of the area under the curve compared by ANOVA).

Figure 14 shows an evaluation of the ability of different immunogens to stimulate inhibition responses of specific NS3 / 4A HCV tumors after a single immunization. Groups of ten C57BL / 6 mice were left untreated or were given an immunization with the indicated immunogen (4 Ig of DNA using a gene gun in (a), (b), (c), (g) and (h); 107 particles of SFV sc in d; 100 Ig peptide in CFA sc in €; and 20 Ig of NS3 in CFA sc in (f) Two weeks after the last immunization, mice were injected subcutaneously with EL-4 cells expressing NS3 / 4A. Tumor sizes were measured through the skin 6 to 19 days after tumor injection. Values have been provided as mean tumor size ± standard error.

€, as a negative control, in each graph the average data of the group immunized with the

empty pVAX plasmid. In (f) to (h) the negative controls were non-immunized mice. Likewise, the p-value obtained from the statistical comparison of the control with each curve is provided using the area under the curve and ANOVA.

Figure 15 shows the comparative efficiency of plasmids wtNS3 / 4A-pVAX1 and coNS3 / 4A-pVAX1 released with a gene gun in stimulating immune responses of tumor inhibition. Groups of ten BALB / c mice were left untreated or were given one, two or three monthly immunizations with 4 Ig plasmid. Two weeks after the last immunization, mice were injected subcutaneously with 106 SP2 / 0 cells expressing NS3 / 4A. Tumor sizes were measured through the skin at 6, 8, 10, 11, 12, 13 and 14 days after tumor injection. Values have been provided as the mean tumor size ± standard error. A "**" sign indicates a statistical difference of p <0.01, a "*" sign indicates a difference of p <0.05 and NS (not significant) indicates absence of statistical difference (the values of the area under the curve compared by ANOVA).

Figure 16 shows the effect of therapeutic vaccination with plasmid coNS3 / 4A using the gene gun. Groups of ten C57BL / 6 mice were inoculated with 106 EL-4 cells expressing NS3 / 4th. A group had been immunized once with 4Ig of coNS3 / 4A DNA using a gene gun twice before exposure (positive control), a group was similarly immunized six days after tumor inoculation and a group was immunized 12 days after tumor inoculation. One group was not immunized (negative control). Tumor sizes were measured through the skin at 6, 10, 11, 12, 13, 14, 18, 19, and 20 days after tumor injection. Values have been provided as the mean tumor size ± standard error. A "**" sign indicates a statistical difference of p <0.01, a "*" sign indicates a difference of p <0.05 and NS (not significant) indicates absence of statistical difference (the values of the area under the curve compared by ANOVA).

Detailed description of the invention

A new nucleic acid and protein corresponding to the NS3 / 4A domain of HCV of a patient infected with HCV (SEQ ID No. 1) was cloned. A search in Genebank revealed that the cloned sequence had the highest homology with the HCV sequences but had a homology of only 93% with the closest relative of the HCV (accession number AJ 278830). This new peptide (SEQ ID NO. 2) and fragments thereof (e.g., SEQ ID. NO. 14 and 15) that are any number of consecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive amino acids in length), nucleic acids encoding these molecules, vectors having said nucleic acids and cells having said vectors, acids Nucleic or peptides are disclosed herein. It was also found that both the NS3 / 4A gene (SEQ ID No. 1) and the corresponding peptide (SEQ ID No. 2) were immunogenic in vivo.

Mutants of the new NS3 / 4A peptide were created. It was found that the truncated mutants (e.g., SEQ ID NOS. 12 and 13) and mutants lacking a proteolytic cleavage site (e.g., SEQ ID. NO. 3-11) were also immunogenic in vivo. These new peptides (SEQ ID NO. 3-13) and fragments thereof (e.g., SEQ ID. NO. 16 and 26) that are any number of consecutive amino acids between at least 3-50 (e.g., 3 , 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive amino acids in length), nucleic acids encoding these molecules, vectors having said nucleic acids and cells having said vectors, nucleic acids or peptides are disclosed herein.

A codon optimized nucleic acid encoding NS3 / 4a was also created and found to be immunogenic. The nucleic acid of SEQ ID No. 1 was analyzed according to the use of codons and the sequence was compared with the codons that are most used in human cells. Since HCV is a human pathogen, it was unexpected to discover that the virus had not yet evolved to the use of codons that most often encode human proteins (eg, optimal human codons). A total of 435 nucleotides were replaced to generate the synthetic NS3 / 4A nucleic acid optimized by codons. The NS3 / 4A peptide encoded by the codon optimized nucleic acid sequence (SEQ ID NO: 36) had a 98% homology with HCV-1 and contained a total of 15 different amino acids.

It was found that codon-encoded nucleic acid (MSLF1 or coNS3 / 4a) SEQ ID No. 35) translated more efficiently in vitro than native NS3 / 4A and that mice immunized with the MSLF1-containing construct generated significantly more specific antibodies of NS3 / 4A than mice immunized with a construct containing wild NS3 / 4A. In addition, mice immunized with the MSLF1-containing construct were found to stimulate NS3-specific CTLs more efficiently and exhibited better immune responses of tumor inhibition in vivo than mice immunized with constructs containing wild NS3 / 4A.

The peptides and nucleic acids described above are useful as immunogens that can be administered alone or together with an adjuvant. Preferred embodiments include compositions comprising one or more of the nucleic acids of the invention described above with or without an adjuvant. That is, some of the compositions described herein are prepared with or without an adjuvant and comprise, consist of

or consist essentially of an NS3 / 4A nucleic acid of the invention. (e.g., SEQ ID No. 35) or a fragment thereof that is any number of consecutive nucleotides between at least 50 (e.g., 50, -100, 100, -200, 200, -500, 500, -1000, 1000, -1500, 1500, -2079, 1500 or 1500-2112 consecutive nucleotides) in length with the condition cited in claim 2.

It was also found that compositions comprising ribavirin and an antigen (eg, one or more of the previously described HCV peptides or nucleic acids) enhance and / or facilitate an animal's immune response to the antigen. That is, it was discovered that ribavirin is a very effective "adjuvant" that, for the purposes of the present disclosure, refers to a material that has the ability to enhance or facilitate an immune response to a particular antigen. The adjuvant activity of ribavirin was manifested by a significant increase in the protection mediated by the immune system against the antigen, an increase in the titer of the antibodies produced against the antigen and an increase in proliferative T cell responses.

Accordingly, compositions (eg, vaccines and other medicaments) comprising ribavirin and one or more of the nucleic acids of the invention are embodiments of the invention. These compositions may vary according to the amount of ribavirin, the form of ribavirin, as well as the HCV nucleic acid sequence as cited in the cited claims.

Embodiments of the invention also include methods of making and using the above compositions. Some procedures described involve the manufacture of nucleic acids encoding NS3 / 4A, NS3 / 4th codon optimized, NS3 / 4A mutants, fragments thereof that are any number of consecutive nucleotides between at least 9-100 (e.g., 9, 12, 15, 18, 21, 24, 27, 30, 50, 60, 75, 80, 90 or 100 consecutive nucleotides in length), peptides corresponding to said nucleic acid constructs comprising said nucleic acids containing said compositions . Some methods described relate to the manufacture of vaccine compositions or immunogenic preparations comprising, consisting of or consisting essentially of the newly discovered fragment of codon-optimized NS3 / 4A, NS3 / 4A or mutant NS3 / 4A (e.g., a truncated mutant or a mutant lacking a proteolytic cleavage site) or a fragment thereof or a nucleic acid encoding one or more of these molecules, as described above. Preferred fragments for use with the methods of the invention include fragments of SEQ ID No. 35 containing at least 50 consecutive nucleotides with the condition cited in the claim.

2. The compositions described above may be manufactured by providing an adjuvant (eg, ribavirin), providing an HCV antigen (eg, a peptide comprising an HCV antigen such as (SEQ ID No. 211 or 36 ) or a fragment thereof such as, SEQ ID No. 12-26 or a nucleic acid encoding one or more of said peptides) and mixing said adjuvant and said antigen to formulate a composition that can be used to enhance or facilitate a immune response in a subject to said antigen.

The nucleic acid of the invention is also provided for use in methods of potentiating or stimulating an immune response in an animal, including humans, to an antigen. Such procedures can be practiced by, for example, the identification of an animal that needs an immune response to HCV and providing said animal with a composition comprising one or more of the above nucleic acids and an amount of adjuvant that is effective for enhance or facilitate an immune response to the antigen / epitope. In some embodiments, the antigen and adjuvant are formulated for administration separately rather than in a single mixture. Preferably, in this case, the adjuvant is formulated for administration a short period of time before or a short period of time after administration of the antigen.

Other embodiments refer to the use of a nucleic acid of the invention to prepare a medicament suitable for treating and preventing HCV infection. By one approach, an immunogen comprising one or more of the HCV acids of the invention is used to prepare a medicament for the treatment and / or prevention of HCV infection. By another approach, an individual is identified as needing a medicament that prevents and / or treats HCV infection and said individual is provided with a medicament comprising ribavirin and an HCV antigen nucleic acid of the invention.

The next section discusses the discovery of the new NS3 / 4A gene, the codon optimized NS3 / 4A gene, the creation of the NS3 / 4A mutants and the characterization of the corresponding nucleic acids and peptides.

NS3 / 4A, NS3 / 4A and NS3 / 4A codon optimized mutants

A new nucleic acid and protein corresponding to the HCV NS3 / 4A domain of a patient infected with HCV (SEQ ID NO. 1 and 2) was cloned. A search in Genebank revealed that the cloned sequence had the highest homology with the HCV sequences but had a homology of only 93% with the closest relative of the HCV (accession number AJ 278830). A truncated mutant of the new NS3 / 4A peptide and NS3 / 4A mutants that lack a proteolytic cleavage site (as well as the corresponding nucleic acids) were also created. In addition, a codon and peptide optimized human NS3 / 4A nucleic acid was created. It was discovered that these new peptides and nucleic acids encoding said peptides were potent immunogens that can be mixed with adjuvants to make a composition that induces a receptor to provide an immune response to HCV. The cloning of the new NS3 / 4A gene and the creation of the various NS3 / 4A mutants and the codon optimized NS3 / 4A gene are described in the following example.

Example 1

The NS3 / 4A sequence was amplified from the serum of a patient infected with HCV (HCV genotype 1a) using the polymerase chain reaction (PCR). Total RNA was extracted from serum and cDNA synthesis and PCR were performed according to conventional protocols (Chen M et al., J. Med. Virol. 43: 223-226 (1995)). The cDNA synthesis was initiated using the "NS4KR" antisense primer (5'-CCG TCT AGA TCA GCA CTC TTC CAT TTC ATC-3 '(SEQ ID NO: 28)). From this cDNA a DNA fragment of 2079 base pairs, corresponding to amino acids 1007 to 1711, which encompasses the NS3 and NS4A genes was amplified. A high fidelity polymerase (Expand High Fidelity PCR, Boehringer-Mannheim, Mannheim, Germany) was used with the primer "NS3KR" (5'-CCT GAA TTC ATG GCG CCT ATC ACG GCC TAT-3 '(SEQ ID No. 29) and the NS4KR primer The NS3KF primer contained an EcoRI restriction enzyme cleavage site and an initiation codon and the NS4KR primer contained an XbaI restriction enzyme cleavage site and a termination codon.

The amplified fragment was then sequenced (SEQ ID NO. 1). Sequence comparison analysis revealed that the gene fragment had been amplified from a viral strain of genotype 1a. A BLAST computerized search against the Genbank database using the NCBI website revealed that the closest HCV counterpart had a 93% identity in the nucleotide sequence.

The amplified DNA fragment was then digested with EcoRI and XbaI and inserted into a plasmid pcDNA3.1 / His (Invitrogen) digested with the same enzymes. Plasmid NS3 / 4A-pcDNA3.1 was then digested with EcoRI and Xba I and the insert was purified using the QiaQuick kit (Qiagen, Hamburg, Germany) and ligated into a pVAX vector digested with EcoRI / Xba I (Invitrogen) generating the NS3 / 4A-pVAX plasmid.

The truncated rNS3 mutant was obtained by deleting the NS4A sequence from the NS3 / 4A DNA. Accordingly, the NS3 gene sequence of NS3 / 4A-pVAX was amplified by PCR using primers NS3KF and 3'NotI (5'-CCA CGC GGC CGC GAC GAC CTA CAG-3 '(SEQ ID No. 30)) which contain the EcoRI and Not I restriction sites respectively. The NS3 fragment (1850 bp) was then ligated into a plasmid pVAX digested with EcoRI and Not I generating the NS3-pVAX vector. Plasmids were cultured in E. coli BL21 cells. Plasmids were sequenced and verified by restriction cleavage and the results were as expected based on the original sequence.

Table 1 describes the sequence of the proteolytic cleavage site of NS3 / 4A, called the cut-off point between NS3 and NS4A. This sequence of the wild cut-off point was mutilated in many different ways in order to generate several different NS3 / 4A cut-off mutants. Table 1 also identifies these mutant cutoff point sequences. The fragments listed in Table 1 are preferred immunogens that can be incorporated with or without an adjuvant (e.g., ribavirin) in a composition by administering to an animal in order to induce an immune response in said animal against HCV.

TABLE 1

Plasmid

Deduced amino acid sequence

* NS3 / 4A-pVAX

TKYMTCMSADLEVVTSTWVLVGGVL (SEQ ID No. 14)

NS3 / 4A-TGT-pVAX

TKYMTCMSADLEVVTGTWVLVGGVL (SEQ ID No. 16)

NS3 / 4A-RGT-pVAX

TKYMTCMSADLEVVRGTWVLVGGVL (SEQ ID No. 17)

NS3 / 4A-TPT-pVAX

TKYMTCMSADLEVVTPTWVLVGGVL (SEQ ID NO. 18)

NS3 / 4A-RPT-pVAX

TKYMTCMSADLEVVRPTWVLVGGVL (SEQ ID NO. 19)

NS3 / 4A-RPA-pVAX

TKYMTCMSADLEVVRPAWVLVGGVL (SEQ ID No. 20)

NS3 / 4A-CST-pVAX

TKYMTCMSADLEVVCSTWVLVGGVL (SEQ ID NO. 21)

NS3 / 4A-CCST-pVAX

TKYMTCMSADLEVCCSTWVLVGGVL (SEQ ID No. 22)

NS3 / 4A-SSST-pVAX

TKYMTCMSADLEVSSSTWVLVGGVL (SEQ ID No. 23)

NS3 / 4A-SSSSCST-pVAX

TKYMTCMSADSSSSCSTWVLVGGVL (SEQ ID No. 24)

NS3A / 4A-VVVVTST-pVAX

TKYMTCMSADVVVVTSTWVLVGGVL (SEQ ID NO. 25)

NS5-pVAX

ASEDVVCCSMSYTWTG (SEQ ID No. 27)

NS5A / B-pVAX

SSEDVVCCSMWVLVGGVL (SEQ ID No. 26)

* The wild sequence for the NS3 / 4A fragment is NS3 / 4A-pVAX. The cut-off point of NS3 / 4A is identified by underlining, in which the P1 position corresponds to the first Thr (T) and the P1 position corresponds to the next amino acid in the NS3 / 4A-pVAX sequence. In the wild NS3 / 4A sequence, the NS3 protease cleaves between

positions P1 and P1 ’.

By changing the proteolytic site between NS3 and NS4A, plasmid NS3 / 4A-pVAX was mutagenized using the QUICKCHANGE ™ (Stratagene) mutagenesis kit, following the manufacturer's recommendations. By generating the 5 "TPT" mutation, for example, the plasmid was amplified using primers 5'-CTGGAGGTCGTCACGCCTACCTGGGTGCTCGTT-3 '(SEQ ID No. 31) and 5'-ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3' as SEQ ID No. 323 which has SEQ ID No. 323 / 4A-TPTpVAX. Generating the "RGT" mutation, for example, the plasmid was amplified using primers 5'-CTGGAGGTCGTCCGCGGTACCTGGGTGCTCGTT-3 '(SEQ ID No. 33) and 5'-ACCGAGCACCCAGGTACC

10 GCGGACGACCTCCAG-3 '(SEQ ID No. 34) resulting in NS3 / 4A-RGT-pVAX. All mutagenized constructs were sequenced verifying that the mutations had been created correctly. Plasmids were cultured in competent E. coli BL21 cells.

The sequence of the NS3 / 4A single gene previously isolated and sequenced (SEQ ID No. 1) was analyzed according to the use of

15 codons with respect to the codons most used in human cells. A total of 435 nucleotides were replaced optimizing the use of codons for human cells. The sequence was sent to Retrogen Inc. (6645 Nancy Ridge Drive, San Diego, CA 92121) and instructions were provided generating an NS3 / 4A gene optimized by synthetic and full length codons. The codon optimized NS3 / 4A gene had a 79% homology within the region between nucleotide positions 3417-5475 of the reference strain VHC-1. A total of 433

20 nucleotides differed. At the amino acid level, the homology with the HCV-1 strain was 98% and a total of 15 amino acids differed.

The 2.1 kb codon optimized and full length HCV DNA fragment corresponding to amino acids 1007 to 1711 spanning the NS3 and NS4A NS3 / NS4A gene fragment was amplified by polymerase chain reaction (PCR). ) using a high-ease polymerase (Expand High Fidelity PCR, Boehringer-Mannheim, Mannheim, Germany). Then, the amplicon was inserted into a pVAX vector digested with Bam HI and Xba I (Invitrogen, San Diego), which generated the plasmid MSLF1-pVAX (coNS3 / 4A-pVAX) All expression constructs were sequenced. Plasmids were grown in E. coli cells. BL21 competent. Plasmid DNA used for in vivo injection was purified using Qiagen DNA purification columns according to

30 manufacturers instructions (Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmid DNA was determined by spectrophotometry (Dynaquant, Pharmacia Biotech, Uppsala, Sweden) and the purified DNA was dissolved in sterile phosphate buffered saline (PS) at concentrations of 1 mg / ml.

The expression of NS3 and NS3 / 4A proteins of plasmids wtNS3 / 4A (wild NS3 / 4A) and coNS3 / 4A are

35 analyzed by an in vitro transcription and translation test. The assay showed that proteins could be correctly translated from plasmids and that plasmid coNS3 / 4A gave detectable NS3 and NS3 / 4A bands at a higher plasmid dilution compared to plasmid wtNS3 / 4th. This result provided strong evidence that in vitro translation of the plasmid coNS3 / 4A is more effective than with wtNS3 / 4A. By comparing the expression levels more accurately, HepG2 cells were transiently transfected with the

40 plasmids wtNS3 / 4A and coNS3 / 4ª. These experiments revealed that plasmid coNS3 / 4A generated expression levels of NS3 protein 11 times higher when compared to plasmid wtNS3 / 4A, as determined by densitometry and a standard curve of recombinant NS3. Since the wtNS3 / 4A and coNS3 / 4A plasmids are identical in size, it is unlikely that there will be any significant difference in the transfection efficiencies between the plasmids. Staining of BHK cells transfected with the coNS3 / 4A plasmid and infected with the SFV revealed a similar perinuclear and cytoplasmic distribution of NS3 as noted above, confirming an unmodified subcellular location.

Using the nucleic acid sequences described above, complementary probes of these molecules can be designed and made by oligonucleotide synthesis. Desirable probes comprise a nucleic acid sequence of (SEQ ID NO: 1) that is unique to this HCV isolate. These probes can be used by screening the cDNA of patients isolating the natural sources of HCV, some of which may themselves be new HCV sequences. Screening can be performed, for example, by hybridization in filters or by PCR. By filter hybridization, the labeled probe preferably contains at least 15-30 base pairs of the nucleic acid sequence of (SEQ ID NO: 1) that is unique to this NS3 / 4A peptide. The hybridization wash conditions used are preferably of medium to high stringency. Hybridization can be performed in 0.5 NaHPO4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 42 ° C overnight and washing can be performed in 0.2X SSC / 0.2% SDS at 42 ° C. For guidance on these conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y .; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience,

N.Y.

HCV nucleic acids can also be isolated from patients infected with HCV using the nucleic acids described herein. (See also Example 1). Accordingly, the RNA obtained from a patient infected with HCV is reverse transcribed and the resulting cDNA is amplified using PCR or other amplification technique. The primers are preferably obtained from the sequence of NS3 / 4A (SEQ ID NO: 1).

For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B.A. Ed. In Methods in Molecular Biology 67: Humana Press, Totowa (1997) and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press). For mRNA amplification, reverse transcription of the RNA within the cDNA followed by PCR (RT-PCR) is within the scope of the invention; or the use of a single enzyme for both stages as described in US Pat. No. 5,322,770. Another technique involves the use of the ligase chain reaction with asymmetric space and reverse transcriptase (RT-AGLCR), as described by Marshall R.L. et al. (PCR Methods and Applications 4: 80-84, 1994).

Briefly, RNA is isolated following conventional procedures. A reverse transcription reaction is performed with the RNA using an oligonucleotide specific primer of the plus 5 ′ end of the amplified fragment as a primer of the first strand synthesis. The resulting RNA / DNA hybrid is "queued" with guanines

using a conventional terminal transferase reaction. Then, the hybrid is digested with RNAse H and the second strand synthesis is primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment are easily isolated. For a review of cloning strategies that can be used, see Sambrook et al., 1989, ant.

In each of these amplification procedures, the primers on either side of the sequence to be amplified are added to a sample of nucleic acid properly prepared together with dNTP and a thermostable polymerase, such as Taq polymerase, Pfu polymerase or Vent polymerase. The nucleic acid in the sample is denatured and the primers specifically hybridize with complementary nucleic acid sequences in the sample. Then, the hybridized primers are extended. Then another cycle of denaturation, hybridization and extension begins. Cycles are repeated many times producing an amplified fragment that contains the nucleic acid sequence between the primer sites. PCR has been further described in several patents, including US patents. 4,683,195. 4,683,202 and 4,965,188.

The primers are selected so that they are substantially complementary to a portion of the nucleic acid sequence of (SEQ ID NO. 1) that is unique to this NS3 / 4A molecule, so that they allow amplification of the sequences between the primers. Preferably, the primers may have a length of any number between at least 16-20, 20-25 or 25-30. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G + C content. The higher the G + C content of the primer, the higher the melting temperature because the G: C pairs are held together by three bridges of H, while the A: T pairs only have two. The G + C content of the amplification primers described herein vary, preferably, between 10% and 75%, more preferably between 35% and 60% and more preferably, between 40% and 55%. One skilled in the art can empirically determine the appropriate length of the primers in a particular set of test conditions.

The spacing of the primers refers to the length of the segment to be amplified. In the context of the embodiments described herein, the size of the amplified segments carrying the nucleic acid sequence encoding the HCV peptides may vary from at least about 25 bp to the full length of the HCV genome. Typical 25-1000 bp amplification fragments are typical, 50-1000 bp fragments are preferred, and 100-600 bp fragments are most preferred. It will be appreciated that the amplification primers can have any sequence that allows specific amplification of the NS3 / 4A region and can include, for example, modifications such as restriction sites facilitating cloning.

The PCR product can be subcloned and sequenced ensuring that the amplified sequences represent the sequences of an HCV peptide. Therefore, the PCR fragment can be used by isolating a full length cDNA clone by several procedures. For example, the amplified fragment can be labeled and used by screening a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment can be used by isolating genomic clones by screening a genomic library. Additionally, an expression library can be constructed using cDNA synthesized from, for example, RNA isolated from an infected patient. Thus, HCV gene products can be isolated using conventional antibody screening techniques together with antibodies produced against the HCV gene product. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor).

Also described are (a) DNA vectors containing any of the above nucleic acid sequences and / or their complementary (ie, antisense); (b) DNA expression vectors containing any of the above nucleic acid sequences operatively associated with a regulatory element that directs nucleic acid expression; and (c) genetically engineered host cells containing any of the above nucleic acid sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. These recombinant constructs are capable of replicating autonomously in a host cell. Alternatively, recombinant constructs can be integrated into the chromosomal DNA of a host cell. Such recombinant polynucleotides typically comprise a genomic HCV or cDNA polynucleotide of semisynthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising these sequences and complementary to them that are not natural are provided.

Although nucleic acids encoding an HCV peptide or nucleic acids having sequences complementary to an HCV gene can be used as they appear in nature, they will often be altered by deletion, substitution or insertion and may be accompanied by sequences not present in Humans. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that direct and regulate expression. Such regulatory elements include, among others, the immediate early hCMV gene, the SV40 adenovirus early or late promoters, the lac system, the trp system, the TAC system, the TRC system, the main operator and the phage promoter regions. A, the control regions of the fd coat protein, the promoter for 3-phosphoglycerate kinase, the acid phosphatase promoters and the yeast mating factor promoters.

In addition, the nucleic acid sequences encoding the recombinant HCV peptide and its complementary sequences can be engineered so that their processing or expression is modified. For example and not by way of limitation, the HCV nucleic acids described herein may be combined with a promoter sequence and / or a ribosome binding site or a signal sequence may be inserted upstream of the peptide coding sequences. HCV in a way that allows peptide secretion and thus facilitates harvesting or bioavailability. Additionally, a given HCV nucleic acid can be mutated in vitro or in vivo creating and / or destroying translation, initiation and / or termination sequences, or creating variations in the coding regions and / or forming new restriction sites or destroying existing ones. , facilitating additional in vitro modifications. (See Example 1). Any technique for mutagenesis known in the art can be used, including, among others, site-directed mutagenesis in vitro. (Hutchinson et al.,

J. Biol. Chem., 253: 6551 (1978)). The nucleic acids encoding the HCV peptides described above can be manipulated using conventional techniques in molecular biology creating the recombinant constructs that express the HCV peptides.

In addition, nucleic acids encoding other proteins or domains of other proteins can bind to nucleic acids encoding an HCV peptide creating a fusion protein. Nucleotides encoding fusion proteins may include, among others, a full-length sequence of NS3 / 4A (SEQ ID No. 2 or SEQ ID No. 36), mutant NS3 / 4A sequences (eg, SEQ ID No. 3-11) or a peptide fragment of an NS3 / 4A sequence condensed with an unrelated protein or peptide, such as, for example, polyhistidine, hemagglutinin, an enzyme, fluorescent protein or luminescent protein, as discussed below.

It has been found that the "NS3 / 4A-pVAX" construct was significantly more immunogenic in vivo than the "NS3-pVAX" construct. Surprisingly, it has also been found that the codon-encoded NS3 / 4A construct (("MSLF1-pVAX") was more immunogenic in vivo than NS3-pVAX. The following example describes these experiments.

Example 2

By determining whether the NS3-pVAX and NS3 / 4A-pVAX vectors elicited a humoral immune response, the expression constructs described in Example 1 were purified using the DNA purification system

5 Qiagen according to the manufacturer's instructions and purified DNA vectors were used by immunizing groups of four to ten Balb / c mice. Plasmids were injected directly into the anterior tibial muscles (TA) in regeneration as described previously (Davis et al., Human Gene Therapy 4 (6): 733 (1993)). In summary, mice were injected intramuscularly with 50 Il / TA of 0.01 mM cardiotoxin (Latoxan, Rosans, France) in sterile 0.9% NaCl. Five days later, 50 IL of PBS containing rNS3 or DNA was injected into each TA muscle.

10 Inbred mouse strains C57 / BL6 (H-2b), Balb / C (H-2d), and CBA (H-2k) were obtained from the breeding center in Möllegard Denmark, Charles River Uppsala, Sweden, or B&K Sollentuna Sweden . All mice were female and were used at 4-8 weeks of age. For monitoring humoral responses, all mice received a 50 Il / TA booster injection of plasmid DNA every four weeks. In addition, it was administered

15 to some mice the recombinant NS3 protein (rNS3), which was purified as described herein. Mice that received rNS3 were not immunized more than twice. Blood was drawn from all mice twice a month.

Enzyme immunosorption assays (EIA) were used to detect the presence of NS3 specific antibodies

20 murine. These assays were performed essentially as described (Chen et al., Hepatology 28 (1): 219 (1998)). Briefly, rNS3 was passively adsorbed overnight at 4 ° C in 96-well microtiter plates (Nunc, Copenhagen, Denmark) at 1 Ig / ml in 50 mM sodium carbonate buffer (pH 9.6). The plates were then blocked by incubation with dilution buffer containing PBS, 2% goat serum and 1% bovine serum albumin, for one hour at 37 ° C. Then serial dilutions of mouse serum starting at

25 1:60 were incubated on the plates for one hour. The bound murine serum antibodies were detected by goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma Cell Products, Saint Louis, MO), followed by the addition of the pNPP substrate (1 tablet / 5 ml of 1M diethanolamine buffer with MgCl2 0.5 mM The reaction was stopped by the addition of 1M NaOH and the absorbance was read at 405 nm.

30 After four weeks, four out of five mice immunized with NS3 / 4A-pVAX had developed antibodies against NS3, while one out of five immunized with NS3-pVAX had developed antibodies (Figure 1). After six weeks, four out of five mice immunized with NS3 / 4A-pVAX had developed high levels ((> 104) of antibodies against NS3 (mean levels 10,800 ± 4,830) and one had a titer of 2,160, although all mice immunized with NS3 / 4A-pVAX developed antibodies against NS3 none of them developed such high levels

35 as produced by the NS3 / 4A-pVAX construction (average levels 1,800 ± 805). The antibody levels caused by the NS3 / 4A fusion construct were significantly higher than those induced by NS3-pVAX at six weeks (mean ranges from 7.6 to 3.4 p <0.05, proof of the sum of Mann-Whitney tangos and p <0.01, Student's t-test, therefore, immunization with NS3-pVAX or NS3 / 4A-pVAX resulted in the production of NS3-specific antibodies, but the construction containing NS3 / 4A It was a more potent immunogen.

40 A similar experiment was performed comparing the immunogenicity of the NS3 / 4A-pVAX and MSLF1pVAX constructs. Simulating better a future vaccination program in humans, plasmids were released in groups of ten mice using a gene gun. In summary, the plasmid DNA was bound to gold particles according to the protocols supplied by the manufacturer ((Bio-Rad Laboratories, Hercules, CA). Before immunization,

The injection area was shaved and immunization was performed according to the manufacturer's protocol. Each injection dose contained 4 Ig of plasmid DNA. Immunizations were performed weeks 0, 4 and 8.

The MSLF1 gene was found to be more immunogenic than the native NS3 / 4A gene, since NS3-specific antibodies were significantly higher in mice immunized with the MSLF1-pVAX construct at two

50 weeks of the second and third immunization (table 2). These results confirmed that the codon optimized MSLF1-pVAX was a more potent B-cell immunogen than NS3 / 4A-pVAX.

TABLE 2

Immunogenic

Week No. of injections NS3 Middle Title SD Mann-Whitney

NS3 / 4A

2 one 0 0 NS

MSLF1

2 one 0 0

NS3 / 4A

6 2 0 0 p <0.0002

MSLF1

6 2 2484 3800

NS3 / 4A

10 3 60 0 p <0.0001

MSLF 1

10 3 4140 4682

55 The following example provides more evidence that MSLF-1 (coNS3 / 4a) produces a strong humoral response. 13

Example 2A

Analyzing the intrinsic immunogenicity of the different groups of BALB / c NS3 genes, mice (H-2d) were immunized with the following vectors. wtNS3 / 4A (NS3 / 4A wild), coNS3 / 4A (NS3 / 4a or MSLF-1 optimized by codon), or wtNS3 / 4A-SFV (wild NS3 / 4A obtained from expression in SFV). Doses of 4 Ig of DNA were administered using the gene gun and doses of 107 SFV particles were injected subcutaneously (s.c.). The mice received booster doses at four weeks. Mice immunized with wtNS3 / 4A-SFV developed antibodies already after the first injection, suggesting a potent immunogenicity (Figure 10). Two weeks after the second immunization, most mice immunized with coNS3 / 4A vectors or

10 wtNS3 / 4A-SFV had developed average antibody levels of more than 103 (Figure 10). In contrast, none of the mice immunized with the plasmid wtNS3 / 4A had developed detectable NS3 specific antibodies at six weeks (Figure 10). Therefore, both codon optimization and amplification of mRNA by SFV result in an increase in the immunogenicity of B lymphocytes of the NS3 / 4A gene.

15 Indirectly comparing the division of T1 helper (Th1) and Th2 lymphocytes of the T lymphocyte response stimulated by immunizations with wtNS3 / 4A, coNS3 / 4A and wtNS3 / 4A-SFV, levels of IgG1 type antibodies were analyzed ( Th2) and of IgG2a (Th1) specific to NS3 (Figure 10). The IgG2a / IgG1 ratio in mice immunized with rNS3 with or without adjuvant was always <1 regardless of the murine haplotype, signaling a Th2-dominated response. In contrast, mice immunized i.m. with plasmids that

20 contain wtNS3 (wild NS3), wtNS3 / 4A, or coNS3 / 4A had Th lymphocyte responses biased to Th1 evidenced by IgG2a / IgG1 ratios of> 1 (Figure 10). Therefore, codon optimization did not change the subclass distribution of IgG. When genetically immunizing BALB / c mice with NS3 / 4A using the gene gun, the subclass ratio suggested a mixed Th1 / Th2 response (Figure 10). It should be noted that the codon-optimized plasmid did not show an increase in the in vitro stimulatory capacity of B cells, in

25 comparison with the native plasmid, which suggests that important immune stimulation motives had not been lost or introduced.

Immunizations using SFV stimulated a distribution of Th1 or mixed Th1 / Th2 isotypes. The IgG2a / IgG1 anti-NS3 ratio after immunization with wtNS3 / 4A-SFV ranged from 2.4 to 20 between different experiments,

30 providing signs of a Th1 response. This is similar to previous experience with SFV vectors in which a subclass distribution of IgG biased to Th1 was observed.

The following example describes experiments that were performed by determining if mutant peptides of NS3 / 4A lacking a proteolytic cleavage site could elicit an immune response to NS3.

35 Example 3

Analyzing whether the enhanced immunogenicity of NS3 / 4A could be attributed solely to the presence of NS4A or if the NS3 / 4A fusion protein also had to be cleaved at the junction of NS3 / 4th, another set of 40 experiments was performed. In a first experiment, the immunogenicity of the NS3-pVAX, NS3 / 4ApVAX and mutant NS3 / 4A constructs in Balb / c mice was compared. The mice were immunized at week 0 as described above and after two weeks, blood was drawn. to all mice and the presence of antibodies against NS3 was determined at a serum dilution of 1:60 (table 3). The blood was taken from the mice again at week 4. As shown in Table 3, all constructs induced an immune response; mutant constructs,

For example, the NS3 / 4A-TGT-pVAX vector was comparable to the NS3-pVAX vector (4/10 vs. 0/10; NS, Fisher's exact test). However, the NS3 / 4A-pVAX vector was more potent than the mutant constructs.

TABLE 3

Weeks since 1st immunization

Number of antibody responders to the respective immunogen after an immunization of 100 Ig i.m

NS3-pVAX

NS3 / 4A-wild pVAX Example of mutant NS3 / 4A-TGT-pVAX

2

0/10 17/20 4/10

20/20

10/10

4

0/10 (2415 ± 3715) (390 ± 639)

(<60)

55%> 103 50%> 102

10%> 104

10%> 103

50 During the chronic phase of infection, HCV replicates in hepatocytes and spreads in the liver. A major factor in the combination of chronic and persistent viral infections is the cell-mediated immune defense system. CD4 + and CD8 + lymphocytes infiltrate the liver during the chronic phase of HCV infection, but are unable to eliminate or prevent liver damage. In addition, persistent HCV infection is associated with the onset of hepatocellular carcinoma (CHC). The following examples describe experiments that were performed by determining whether the NS3, NS3 / 4A, and MSLF1 constructs could elicit an immune response mediated by T lymphocytes to NS3.

Example 4

5 Studying whether the constructs described above could elicit a cell-mediated response against NS3, a tumor growth test was performed in vivo. To this end, a tumor cell line SP2 / 0 (myeloma cell line SP2 / 0-Ag14 (H-2d)) stably transfected with the NS3 / 4th gene was created. SP2 / 0 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FCS, Sigma Chemicals, St.

10 Louis, MO), 2 mM L-Glutamine, 10 mM HEPES, 100 U / ml penicillin and 100 Ig / ml streptomycin, 1 mM non-essential amino acids, 50 IM 1-mercaptoethanol, 1mM sodium pyruvate (GIBCO-BRL , Gaithesburgh, MD). Plasmid pcDNA3.1 containing the NS3 / 4A gene was linearized by digestion with BglII. A total of 5 Ig of linearized plasmid DNA was mixed with 60 Ig of the transfection reagent (Superfect, Qiagen, Germany) and the mixture was added to a 50% confluent layer of SP2 / 0 cells in a 35 mm plate. The procedure of

15 transfection was performed according to the manufacturer's protocol.

Transfected cells were cloned by limit dilution and selected by the addition of 800 Ig of geneticin (G418) / ml of complete DMEM medium after 14 days. A stable clone of SP2 / 0 expressing NS3 / 4A was identified using PCR and RTPCR and / or a capture EIA using a monoclonal antibody against NS3. All EIA 20 for the detection of antibodies against murine NS3 were performed essentially as follows. In summary, rNS3 (recombinant NS3 protein produced in E. Coli, dialyzed overnight against PBS and sterilized by filtration) was passively adsorbed overnight at 4 ° C in 96-well microtiter plates (Nunc, Copenhagen, Denmark) at 1 Ig / ml in 50 mM sodium carbonate buffer (pH 9.6). The plates were then blocked by incubation with dilution buffer containing PBS, 2% goat serum and 1% bovine serum albumin, for one hour at 37 ° C. Then serial dilutions of mouse serum starting at

1:60 were incubated on the plates for one hour. The bound murine serum antibodies were detected by goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma Cell Products, Saint Louis, MO), followed by the addition of the pNPP substrate (1 tablet / 5 ml of 1M diethanolamine buffer with MgCl2 0.5 mM The reaction was stopped by the addition of 1M NaOH, then the absorbance at 405 nm was measured.

The growth kinetics in vivo in the SP2 / 0 and NS3 / 4A-SP2 / 0 cell lines were then evaluated in Balb / c mice. Mice were injected subcutaneously with 2 x 106 tumor cells in the right flank. Each day the size of the tumor through the skin was determined. The growth kinetics of the two cell lines was comparable. The average tumor sizes did not differ between the two cell lines at any point, by

35 example. (See table 4).

TABLE 4

Mouse ID

Tumor cell line Maximum tumor size in vivo at the indicated time point

5

6 7 8 eleven 12 13 14 fifteen

one

SP2 / 0 1.6 2.5 4,5 6.0 10.0 10.5 11.0 12.0 12.0

2

SP2 / 0 1.0 1.0 2.0 3.0 7.5 7.5 8.0 11.5 11.5

3

SP2 / 0 2.0 5.0 7.5 8.0 11.0 11.5 12.0 12.0 13.0

4

SP2 / 0 4.0 7.0 8.0 10.0 13.0 15.0 16.5 16.5 17.0

5

SP2 / 0 1.0 1.0 3.0 4.0 5.0 6.0 6.0 6.0 7.0

Group average

1.92 3.3 5.0 6.2 9.3 10.1 10.7 11.6 12.1

6

NS3 / 4A-SP2 / 0 1.0 2.0 3.0 3.5 4.0 5.5 6.0 7.0 8.0

7

NS3 / 4A-SP2 / 0 2.0 2.5 3.0 5.0 7.0 9.0 9.5 9.5 11.0

8

NS3 / 4A-SP2 / 0 1.0 2.0 3.5 3.5 9.5 11.0 12.0 14.0 14.0

9

NS3 / 4A-SP2 / 0 1.0 1.0 2.0 6.0 11.5 13.0 14.5 16.0 18.0

10

NS3 / 4A-SP2 / 0 3.5 6.0 7.0 10.5 15.0 15.0 15.0 15.5 20.0

Group average

1.7 2.7 3.7 5.7 9.4 10.7 11.4 12.4 14.2

P-value of the comparison by Student's t-test between the means of the groups

0.7736 0.6918 0.4027 0.7903 0.9670 0,7986 0.7927 0.7508 0.4623

The following example describes experiments that were performed by determining whether mice immunized with the NS3 / 4A constructs had developed a T-cell response against NS3.

Example 5

Examining whether a T-cell response had been produced by immunization with NS3 / 4A, the ability of the immune defense system of the immunized mouse was analyzed by attacking the tumor cell line expressing 5 NS3. The protocol analyzing the in vivo inhibition of tumor growth of the myeloma cell line SP2 / 0 in Balb / c mice has been described in detail above (Encke et al., J. Immunol. 161: 4917 (1998)). Tumor growth inhibition in this model depends on the stimulation of cytotoxic T lymphocytes (CTL). In a first set of experiments, groups of ten i.m. mice were immunized. five times at one month intervals with 100 Ig of NS3-pVAX or 100 Ig of NS3 / 4A-pVAX. Two weeks after the last immunization,

10 injected 2 x 106 SP2 / 0 or NS3 / 4A-SP2 / 0 cells into the right flank of each mouse. Two weeks later the mice were sacrificed and the maximum tumor sizes were measured. There was no difference between the average tumor sizes in SP2 / 0 and NS3 / 4A-SP2 / 0 in mice immunized with NS3-pVAX. (See table 5).

TABLE 5

Mouse ID

Immunogenic Dose (Ig) Tumor cell line Tumor growth Maximum tumor size (mm)

one

NS3-pVAX 100 SP2 / 0 Yes 5

2

NS3-pVAX 100 SP2 / 0 Yes fifteen

3

NS3-pVAX 100 SP2 / 0 Do not -

4

NS3-pVAX 100 SP2 / 0 Yes 6

5

NS3-pVAX 100 SP2 / 0 Yes 13

Group Total

4/5 9.75 ± 4,992

6

NS3-pVAX 100 NS3 / 4A-SP2 / 0 Yes 9

7

NS3-pVAX 100 NS3 / 4A-SP2 / 0 Yes 8

8

NS3-pVAX 100 NS3 / 4A-SP2 / 0 Yes 7

9

NS3-pVAX 100 NS3 / 4A-SP2 / 0 Do not -

10

NS3-pVAX 100 NS3 / 4A-SP2 / 0 Do not -

3/5

8.00 ± 1.00

Note: Statistical analysis (StatView): Student's t-test with maximum tumor size. P values <0.05 were considered significant. Unpaired t-test for maximum diameter.

Clustering variable: Column 1 Hypothetized difference = 0 Row exclusion: NS3DNA-Tumor-001213 Group information for maximum diameter Group variable: Column 1 Row exclusion: NS3DNA-Tumor-001213

Analyzing whether the administration of different compositions containing NS3 affected the production of a cell-mediated immune response, mice were immunized with PBS, rNS3, a control DNA or the construction of NS3 / 4A and cell sizes were determined, such as have been described above. The construction of NS3 / 4A could produce a sufficient T-cell response to produce a reduction

30 statistically significant in tumor size (See table 6).

TABLE 6 (continued)

Mouse ID

Immunogenic Dose (Ig) Tumor cell line Anti-NS3 Tumor growth of the Maximum tumor size (mm)

one

NS3-pVAX 10 NS3 / 4A-SP210 <60 + 12.0

2

NS3-pVAX 10 NS3 / 4A-SP2 / 0 <60 + 20.0

3

NS3-pVAX 10 NS3 / 4A-SP2 / 0 60 + 18.0

4

NS3-pVAX 10 NS3 / 4A-SP2 / 0 <60 + 13.0

5

NS3-pVAX 10 NS3 / 4A-SP2 / 0 <60 + 17.0

Group average

60 5/5 16.0 ± 3.391

6

NS3-pVAX 100 NS3 / 4A-SP2 / 0 2160 + 10.0

7

NS3-pVAX 100 NS3 / 4A-SP2 / 0 <60 - -

8

NS3-pVAX 100 NS3 / 4A-SP2 / 0 <60 - -

9

NS3-pVAX 100 NS3 / 4A-SP2 / 0 360 - -

10

NS3-pVAX 100 NS3 / 4A-SP2 / 0 <60 + 12.5

Group average

1260 2/5 11.25 ± 1,768

eleven

NS3 / 4A-pVAX 10 NS3 / 4A-SP2 / 0 <60 + 10.0

12

NS3 / 4A-pVAX 10 NS3 / 4A-SP2 / 0 <60 - -

13

NS3 / 4A-pVAX 10 NS3 / 4A-SP2 / 0 <60 - -

14

NS3 / 4A-pVAX 10 NS3 / 4A-SP2 / 0 <60 + 13.0

fifteen

NS3 / 4A-pVAX 10 NS3 / 4A-SP2 / 0 <60 + 13.5

Group average

<60 3/5 12,167 ± 1,893

16

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 60 + 10.0

17

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 360 - -

18

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 2160 + 8.0

19

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 2160 + 12.0

twenty

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 2160 + 7.0

Group average

1380 4/5 9.25 ± 2.217

36

p17-pcDNA3 100 NS3 / 4A-SP2 / 0 <60 + 20.0

37

p17-pcDNA3 100 NS3 / 4A-SP2 / 0 <60 + 7.0

38

p17-pcDNA3 100 NS3 / 4A-SP2 / 0 <60 + 11.0

39

p17-pcDNA3 100 NS3 / 4A-SP2 / 0 <60 + 15.0

40

p17-pcDNA3 100 NS3 / 4A-SP2 / 0 <60 + 18.0

Group average

<60 5/5 14.20 ± 5.263

41

rNS3 / CFA twenty NS3 / 4A-SP2 / 0 > 466560 + 13.0

42

rNS3 / CFA twenty NS3 / 4A-SP2 / 0 > 466560 - -

43

rNS3 / CFA twenty NS3 / 4A-SP2 / 0 > 466560 + 3.5

44

rNS3 / CFA twenty NS3 / 4A-SP2 / 0 > 466560 + 22.0

Four. Five

rNS3 / CFA twenty NS3 / 4A-SP2 / 0 > 466560 + 17.0

Group average

466560 4/5 17,333 ± 4,509

46

PBS - NS3 / 4A-SP2 / 0 <60 + 10.0

47

PBS - NS3 / 4A-SP2 / 0 <60 + 16.5

48

PBS - NS3 / 4A-SP2 / 0 60 + 15.0

49

PBS - NS3 / 4A-SP2 / 0 <60 + 21.0

The following example describes more experiments that were performed by determining whether the reduction in tumor size can be attributed to the generation of NS3-specific T lymphocytes.

5 Example 6

In the following set of experiments, growth inhibition of SP2 / 0 or NS3 / 4A-SP2 / 0 tumors in Balb / c mice immunized with NS3 / 4A-pVAX was evaluated again. In mice immunized with the NS3 / 4A-pVAX plasmid, the growth of NS3 / 4A-SP2 / 0 tumor cells was significantly inhibited compared to

10 the growth of non-transfected SP2 / 0 cells. (See table 7). Therefore, immunization with NS3 / 4A-pVAX produces CTLs that inhibit the growth of cells expressing NS3 / 4A in vivo.

TABLE 7

Mouse ID

Immunogenic Dose (Ig) Tumor cell line Tumor growth Maximum tumor size (mm)

eleven

NS3 / 4A-pVAX 100 SP2 / 0 Do not -

12

NS3 / 4A-pVAX 100 SP2 / 0 Yes 24

13

NS3 / 4A-pVAX 100 SP2 / 0 Yes 9

14

NS3 / 4A-pVAX 100 SP2 / 0 Yes eleven

fifteen

NS3 / 4A-pVAX 100 SP2 / 0 Yes 25

4/5

17.25 ± 8.421

16

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 Do not -

17

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 Yes 9

18

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 Yes 7

19

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 Yes 5

twenty

NS3 / 4A-pVAX 100 NS3 / 4A-SP2 / 0 Yes 4

4/5

6.25 ± 2.217

Note: Statistical analysis (StatView): Student's t-test with maximum tumor size; P values <0.05 are considered significant.

Unpaired t-test for maximum diameter. Clustering variable: Column 1 Hypothetized difference = 0 Row exclusion: NS3DNA-Tumor-001213

Group information for maximum diameter 10 Group variable: Column 1 Row exclusion: NS3DNA-Tumor-001213

In another set of experiments, growth inhibition of SP2 / 0 tumors expressing NS3 / 4A was evaluated.

15 in Balb / c mice immunized with MSLF1-pVAX. In summary, groups of mice were immunized with different immunogens (4 Ig plasmid) using a gene gun on weeks zero, four, eight, twelve and sixteen. Two weeks after the last immunization s.c. approximately 2 x 106 SP2 / 0 cells expressing NS3 / 4A on the right flank of the mouse. The kinetics of tumor growth were monitored by measuring the size of the tumor through the skin on days felt, 11 and 13. The average tumor sizes were calculated and the

20 groups were compared using the non-parametric Mann-Whitney test. On day 14 all mice were sacrificed.

After a single immunization, tumor inhibition responses were observed. (See figure 2 and table 8). After two immunizations, the two plasmids NS3 / 4A-pVAX and MSLF1-pVAX stimulated the tumor inhibition responses. (See figure 3 and table 9). However, the tumors were significantly sized

25 lower in mice immunized with the MSLF1 gene, compared to the native NS3 / 4A gene. After three injections, both plasmids effectively stimulated comparable tumor inhibition responses. (See figure 4 and table 10). These experiments provided signs that the MSLF-1 gene was more efficient in activating immune responses of tumor inhibition in vivo than NS3 / 4A-pVAX.

30 TABLE 8

Group

MSLF1-pVAX1 NS3 / 4A-pVAX1 Not immunized

MSLF1-pVAX1

- N.S. p <0.05

NS3 / 4A-pVAX1

N.S. - p <0.05

Not immunized

p <0.05 p <0.05 -

TABLE 9

Group

MSLF1-pVAX1 NS3 / 4A-pVAX1 Not immunized

MSLF1-pVAX1

- p <0.05 p <0.01

NS3 / 4A-pVAX1

P <0.05 - p <0.01

Not immunized

p <0.01 p <0.01 -

TABLE 10

Group

MSLF1-pVAX1 NS3 / 4A-pVAX1 Not immunized

MSLF1-pVAX1

- N.S. D <0.01

NS3 / 4A-pVAX1

N.S. - P <0.01

Not immunized

P <0.01 p <0.01 -

5 The following example describes experiments that were performed analyzing the efficiency of various compositions containing NS3 in generating a cell-mediated response against NS3.

Example 7

10 Determining whether NS3 specific T cells were produced by NS3 / 4A immunizations, a tumor cell lysis assay mediated by T cells was used in vitro. The assay has been described in detail above (Sallberg et al., J. Virol. 71: 5295 (1997)). In a first set of experiments, groups of five Balb / c mice were immunized three times with 100 Ig of NS3 / 4A-pVAX i.m. Two weeks after the last

The injection was sacrificed to the mice and the splenocytes were collected. Stimulation cultures were established with 3 x 106 splenocytes and 3 x 106 NS3 / 4A-SP2 / 0 cells. After five days a standard Cr51 release assay was performed using NS3 / 4A-SP2 / 0 or SP2 / 0 cells as targets. The percentage of specific lysis was calculated as the ratio between lysis of NS3 / 4A-SP2 / 0 cells and lysis of SP2 / 0 cells. Mice immunized with NS3 / 4A-pVAX showed specific lysis above 10% in four of five mice analyzed using a

20 effector ratio: target of 20: 1 (See Figures 5A and 5B).

In the following set of experiments, the T cell responses to MSLF1-pVAX and NS3 / 4ApVAX were compared. The ability of the two plasmids to stimulate detectable CTL in vitro was evaluated in C57 / BL6 mice, since an H3-restricted NS3 epitope had previously been mapped. The groups of mice were immunized

25 with the two plasmids and CTLs were detected in vitro using peptide-coated H-2b cells expressing RMS-S or EL-4 cells expressing NS3 / 4A. In summary, in vitro stimulation was carried out for five days in 25 ml flasks at a final volume of 12 ml, containing 5 U / ml of recombinant IL-2murin (mIL-2; R&D Systems, Minneapolis, MN) . The restyling culture contained a total of 40 x 106 immune splenocytes and 2 x 106 irradiated syngeneic SP2 / 0 cells (10,000 rad) expressing the NS3 / 4th proteins. After five days it was done

30 in vitro stimulation by conventional 51 Cr release assay. Effector cells were collected and a four hour 51 Cr test was performed on 96-well U-bottom plates in a total volume of 200 Il. A total of 1 x 10 6 target cells were labeled for one hour with 20 Il of 51 Cr (5 mCi / ml) and then washed three times in PBS. Cytotoxic activity was determined at proportions between effector cells: target (E: D) of 40: 1, 20: 1 and 10: 1, using 5 x 103 target cells labeled with 51 Cr / well.

Alternatively, the splenocytes of C57BL / 6 mice were collected 12 days after peptide immunization and resuspended in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-Glutamine, 10 mM HEPES, 100 U / ml of penicillin and 100 Ig / ml streptomycin, 1mM of non-essential amino acids, 1-mercaptoethanol 50 IM, 1mM sodium pyruvate. In Vitro stimulation was carried out for five days in 25 ml flasks in a

40 total volume of 12 ml containing 25 x 106 splenocytes and 25 x 106 irradiated syngeneic splenocytes (2,000 rad). Restyling was performed in the presence of the NS3 / 4A H-2Db 0.05 IM binding peptide (GAVQNEVTL sequence SEQ ID No. 37) or a control peptide of the H-2Db peptide (KAVYNFATM sequence SEQ ID No. 38). After five days a 51 Cr release test was performed. The RMA-S target cells received pulses with 50IM peptide for 1.5 hours at 37 ° C before labeling with 51 Cr and then washed three times with PBS. The cells

45 effectors were collected and the four hour 51 Cr test was performed as described. Cytotoxic activity was determined at proportions of E: D 60: 1, 20: 1 and 7: 1 with 5 x 103 target cells labeled with 51 Cr / well. These tests determined that the MSLF1 gene stimulated higher levels of lytic activity in vitro compared to the NS3 / 4A-pVAX vector. (See Figure 6A-6L). Similar results were obtained with the H-2b cells coated with the peptide expressing RMA-S and EL-4 cells expressing NS3 / 4A.

50 Using flow cytometry, additional evidence was obtained that the codon optimized MSLF1 gene stimulated NS3-specific CTLs more effectively than the native NS3 / 4A gene. The frequency of specific CD8 + T cells of the NS3 / 4A peptide was analyzed by ex-vivo staining of splenocytes from mice immunized with NS3 / 4A DNA with the H-2Db fusion protein: recombinant soluble dimeric mouse Ig. Many of the

55 monoclonal antibodies and MHC: Ig fusion proteins described herein were purchased from BDB Pharmingen (San Diego, CA); Anti-CD16 / CD32 (Fc-block ™, clone 2.4G2), anti-CD8 conjugated with FITC (clone 53-6.7), anti-H-2Kb conjugated with FITC (clone AF6-88.5), anti-H-2Db conjugated with FITC (clone KH95), H-2Db: recombinant soluble dimeric mouse Ig, rat-a mouse IgG1 conjugated to PE (clone X56).

Approximately 2x106 splenocytes resuspended in 100 Il of PBS / 1% FCS (buffer for FACS) were incubated with 1 Ig / 106 Fc blocking antibody cells on ice for 15 minutes. The cells were then incubated on ice for 1.5 hours with 2 Ig / 106 H-2Db: Ig cells preloaded for 48 hours at + 4 ° C with an excess of 640 nM of NS3 / 4A derived peptide (sequence GAVQNEVTL SEQ ID NO. 37) or 2 Ig / 106 H-2Db fusion protein cells: Ig unloaded. The cells were then washed twice in FACS buffer and resuspended in 100 µl of FACS buffer containing 10 µl / 100 µl of rat-a mouse conjugated secondary mouse IgG1 antibody and incubated on ice for 30 minutes. Then, the cells were washed twice in FACS buffer and incubated with 1Ig / 106 FITC conjugated a-mouse CD8 antibody cells for 30 minutes. Then, the cells were washed twice with FACS buffer and resuspended in 0.5 ml of FACS buffer containing 0m5 Ig / ml of PI. Approximately 200,000 events from each sample were acquired in a FACS Calibur (BDB) and dead cells (PI-positive cells) were excluded from the analysis.

The advantage of quantifying specific CTLs by FACS analysis is that it overcomes the possible disadvantages of in vitro expansion of in vitro CTLs before analysis. Direct ex-vivo quantification of NS3-specific CTLs using divalent H-2Db: Ig fusion protein molecules loaded with the NS3 peptide revealed that the codon-optimized MSLF-1 gene effectively stimulated NS3-specific CTLs after two immunizations, while the original NS3 / 4A gene did not (table). Therefore, the optimized MSLF-1 gene effectively stimulates NS3-specific CTLs that are more frequent and have better functionality by all parameters analyzed, compared to the original NS3 / 4A gene. The following example provides more signs that codon optimized NS3 / 4A efficiently stimulates NS3 specific cytotoxic T cells.

Example 7A

Initially, the frequency of NS2-specific CTLs that could be stimulated by gene gun immunization was determined using plasmids expressing wtNS3, wtNS3 / 4A and coNS3 / 4th. Plasmid coNS3 / 4A estimated the higher precursor frequencies of NS3-specific CTLs compared to the wtNS3 gene, which reinforces the importance of NS4A (Figure 11). No statistical differences in CTL precursor frequencies were observed between plasmids expressing wtNS3 / 4A and coNS3 / 4A when analyzed directly ex vivo (Figure 11). A single immunization with the plasmid coNS3 / 4A or wtNS3 / 4A-SFV stimulated about 1% of peptide-specific CTLs in the two weeks after immunization (Figure 11). The specificity of the detection of NSL-specific CTLs was confirmed by a five-day in vitro re-stipulation with the NS3 peptide whereby high frequencies of the precursor were observed after immunization with the coNS3 / 4A gene (Figure 11).

Directly comparing the in vitro lytic activity of the NSL-specific CTLs by the different vectors, a conventional 51 Cr release assay was performed after one or two immunizations. The lytic activity of CTL stimulated in vivo was evaluated in RMA-S cells expressing H-2Db loaded with the NS3 peptide and EL-4 cells stably expressing NS3 / 4A. After one dose, the coNS3 / 4A plasmid and the wtNS3 / 4A-SFV vector was clearly more efficient than the wtNS3 / 4A plasmid in CTL stimulation that lysed the target cells coated with the NS3 peptide (Figure 12). Therefore, the CTL stimulation event was enhanced by codon optimization or RNA amplification of the NS3 / 4A gene. The difference was less clear when using EL-4 cells expressing NS3 / 4th probably because this assay is less sensitive (Figure 12). After two immunizations, all NS3 / 4A vectors appeared to stimulate NS3-specific CTLs with similar efficiency (Figure 12). However, two immunizations with any of the vectors containing NS3 / 4A were clearly more efficient in stimulating NSL-specific CTLs compared to the plasmid containing only the wtNS3 gene (Figure 12), which is completely consistent with the analysis. of the precursor of the CTL and the previous observations. Thus, codon optimization or RNA amplification stimulates NS3-specific CTLs more rapidly.

Experts in the field recognize that analysis of tumor growth inhibition in vivo in BALB / c mice using SP2 / 0 myeloma cells or in C57BL / 6 mice using EL-4 lymphoma cells expressing a HCV viral antigen represent the specific immune response of functional HCV in vivo. (See Encke J et al., J Immunol 161: 4917-4923 (1998)). An SP2 / 0 cell line stably expressing NS3 / 4A has been previously described (see, Frelin L et al., Gene Ther 10: 686-699 (2003)) and an EL-4 cell line expressing NS3 / 4A It was characterized as described below.

Confirming that tumor growth inhibition using the EL-4 cell line expressing NS3 / 4th is completely dependent on a specific immune response of NS3 / 4A a control experiment was performed. Groups of ten C57BL / 6 mice were left unimmunized or immunized twice with plasmid NS3 / 4A. Two weeks after the last immunization the mice were exposed to a s.c. of 106 native EL-4 cells

or EL-4 cells expressing NS3 / 4A (NS3 / 4A-EL-4). For protection a specific immune response of NS3 / 4A was required, since only the immunized mice were protected against the growth of the NS3 / 4A-EL-4 cell line (Figure 13). Therefore, this restricted model of H-2b behaves in a similar way to the restricted model of SP2 / 0 H-2d.

Immunizations with recombinant NS3 protein provided evidence that NS3 / 4th specific B cells and CD4 + T cells were not of fundamental importance in protecting against tumor growth. In vitro depletion of CD4 + or CD8 + T cells from splenocytes of H-2b mice immunized with the plasmid coNS3 / 4A provided evidence that CD8 + T cells were the main effector cells in the 51 Cr release assay. Defining the population of functional antitumor effector cells in vivo, CD4 + or CD8 + T cells were selectively depleted in mice immunized with plasmid coNS3 / 4A one week before and during, exposure with EL-4 cell line expressing NS3 / 4A. Flow cytometric analysis revealed that 75% of the CD4 + and CD8 + T cells had been depleted, respectively. This experiment revealed that in vivo depletion of CD4 + T cells did not have a significant effect on tumor immunity (Figure 13). In contrast, depletion of CD8 + T cells in vivo significantly reduced tumor immunity (p <0.05, ANOVA; Figure 13). Therefore, as expected, NS3 / 4A-specific CD8 + CTLs appear to be the main protective cells in the effector stage in the in vivo model for tumor growth inhibition.

The tumor exposure model was then used to evaluate the efficacy of different immunogens in stimulating a protective immunity against the growth of NS3 / 4A-EL-4 tumor cells in vivo. Ensuring that the effectiveness of the stimulation event was studied, all mice were immunized only once. Fully consistent with in vitro CTL data, the authors of the present invention found that only vectors containing NS3 / 4A could rapidly stimulate protective immune responses compared to those immunized with the empty pVAX plasmid (p <0.05, ANOVA ; figure 14). However, this depended on NS4A but was independent of codon optimization or mRNA amplification, suggesting that C57BL / 6 mice are fairly easily protected against tumor growth using genetic immunization.

Further clarifying the above requirements for stimulation of the protective responses of CD8 + CTL additional experiments were performed. First, C57BL / 6 mice immunized with the CT3 peptide derived from NS3 were not protected against the growth of NS3 / 4A-EL-4 tumors (Figure 14). Second, immunization with recombinant NS3 in adjuvant did not protect against tumor growth (Figure 14). The CT3 peptide derived from NS3 effectively stimulates CTLs in C57BL / 6 and rNS3 mice in adjuvant stimulates high levels of NS3-specific T-helper cells. Therefore, an endogenous production of NS3 / 4A seems to be necessary by stimulating protective CTL in vivo. Further characterizing the stimulation event, the B-cell groups (IMT) or C57BL / 6 mice deficient in CD4 were once immunized with the coNS3 / 4A gene using the gene gun and were exposed two weeks later (Figure 14). Since both lineages were protected against tumor growth, the authors of the invention concluded that neither B cells nor CD4 + T cells were necessary for stimulation of functional NS3 / 4th functional CTLs in vivo (Figure 14). In conclusion, stimulation of protective NS3 / 4A specific CTLs against tumors in vivo in C57BL / 6 mice requires NS4A and endogenous expression of the immunogen. In C57BL / 6 mice, stimulation is less dependent on the gene release pathway or helper cells, such as B cells or CD4 + T cells. The fact that the stimulation of functional CTLs in vivo by the coNS3 / 4A DNA plasmid was independent of CD4 + helper T cells can help explain the rate at which the stimulation occurred.

Repeated experiments in C57BL / 6 mice using the NS3 / 4A-EL-4 cell line have shown that protection against tumor growth is obtained after the first immunization with the NS3 / 4A gene, independently of the optimization by codons or mRNA amplification. Likewise, after two injections, immunity against the growth of the NS3 / 4A-EL-4 tumor was further enhanced, but only when there was NS4A. Therefore, this model may not be sensitive enough revealing subtle differences in the intrinsic immunogenicity of different immunogens.

Comparing better the immunogenicity of the plasmids of DNA wtNS3 / 4A and coNS3 / 4A additional experiments were performed in H-2d mice and it seemed that at least two immunizations were necessary to obtain a protective immunity against the tumor. It is important to remember that the subclass distribution of IgG obtained after gene gun immunization with the NS3 / 4A gene in BALB / c mice suggested a mixed Th1 / Th2 type response. Therefore, it was possible that the Th2-type immunization pathway (gene gun) in the Th2-prone BALB / c mouse strain could alter capacity by stimulating effective CTL responses in vivo.

Groups of ten BALB / c mice were immunized once, twice or three times with 4 Ig of the respective DNA plasmid using the gene gun (Figure 15). The mice were exposed two weeks after the last injection. Accordingly, these experiments provided more signs that the plasmid coNS3 / 4A stimulated immunity of inhibition of the functional NS3 / 4A specific tumor in vivo more rapidly than the wild plasmid (Figure 15). Two doses of coNS3 / 4A stimulated a significantly better NS3 / 4A specific tumor inhibition immunity compared to the wtNS3 / 4A plasmid (p <0.05, ANOVA; Figure 15). After three doses the tumor inhibition immunity was the same. Therefore, the previous data verified that codon optimization of the NS3 / 4A gene stimulates NS3-specific CTLs more rapidly.

As indicated herein, the NS3 / 4A gene can be used as a vaccine. Although it had been determined that NS3 / 4A rapidly stimulated functional CTLs in vivo, the effect of therapeutic immunization after injection of tumor cells was then analyzed. Groups of ten C57BL / 6 mice were exposed to 106 NS3 / 4A-EL-4 tumor cells. One group was immunized transdermally with 4Ig of coNS3 / 4A at six days and another group at 12 days of tumor exposure. After therapeutic vaccination, both groups had significantly smaller tumors compared to the non-immunized control group (p <0.01, respectively, ANOVA; Figure 16). This confirms that the vaccine rapidly stimulates CTLs that are capable of reaching and infiltrating tumors that express NS3 / 4th. Therefore, gene gun immunization with the plasmid coNS3 / 4A also functions as a therapeutic vaccine. That is, gene gun immunization using the coNS3 / 4A gene six to 12 days after inoculation of tumor cells expressing NS3 / 4th significantly inhibited tumor growth. In general, rapid immunization of specific HCV NS3 immune responses that are functional in vivo are generated by DNA-based immunization with a codon optimized gene or by mRNA amplification by SFV replicon. Using these approaches, one can prepare very effective vaccines for the treatment and prevention of chronic HCV infections. The following example described in more detail some of the materials and procedures used in the experiments described herein.

Example 7B

Mice

Inbred mice BALB / c (H-2d) and C57BL / 6 mice (H-2b) were obtained from commercial suppliers (Möllegard, Denmark). B-cell deficient mice (IMT) were kindly provided by Dr. Karin Sandstedt, Karolinska Institutet, Sweden. C57BL / 6 mice deficient in CD4 were obtained from the breeding center at the Microbiology and Tumor Biology Center, Karolinska Institutet. All mice were female and were used at 4-8 weeks of age at the beginning of the experiments.

Recombinant NS3 protein with the ATPase / helicase domain

The recombinant NS3 protein (rNS3) was kindly provided by Darrell L. Peterson, Department of Biochemistry, Commonwealth University, VA. The production of recombinant NS3 protein (does not include NS4A) in

E. Coli has been described in the field. Before use, the rNS3 protein was dialyzed overnight against PBS and sterilized by filtration.

Generation of a codon optimized synthetic NS3 / 4A (co) gene

The sequence of the single wtNS3 / 4A gene previously isolated and sequenced was analyzed according to the use of codons with respect to the codons most used in human cells. A total of 435 nucleotides were replaced optimizing the use of codons for human cells. The sequence was sent to Retrogen Inc (San Diego, CA) for the generation of a full-length synthetic coNS3 / 4A gene. The NS3 / 4A gene had a sequence homology of 79% within the region at nucleotide positions 3417-5475 of the reference strain VHC-1. A total of 433 nucleotides differed. At the amino acid level, the homology with the HCV-1 strain was 98% (15 amino acids differed).

The full length codon optimized the 2.1 kb DNA fragment of HCV genotype 1b corresponding to amino acids comprising NS3 and NS4A. The NS3 / NS4A gene fragment was inserted into a pVAX vector digested with Bam HI and Xba I (Invitrogen, San Diego), giving plasmid coNS3 / 4A-pVAX. The expression construction was sequenced guaranteeing the correct sequence and reading frame. Protein expression was analyzed by an in vitro transcription and translation assay. Plasmids were cultured in competent E. coli TOP10 cells (Invitrogen). Plasmid DNA used for in vivo injection was purified using Qiagen DNA purification columns according to the manufacturers instructions (Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmid DNA was determined spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala, Sweden). The purified DNA was dissolved in sterile phosphate buffered saline (PBS) at a concentration of 1 mg / ml.

In vitro translation tests

Ensuring that the wtNS3 / 4A and coNS3 / 4A genes were intact and could be translated, an in vitro transcription assay was performed using the T7 reticulocyte lysate system coupled to prokaryotes (TNT; Promega, Madison, WI). Comparing the translation efficiency of the two plasmids, the amount of input DNA was diluted in serial dilutions (from 6 ng to 1 ng) before addition to the TNT assay.

Transient transfections

HepG2 cells were transfected transiently using conventional protocols. In summary, HepG2 cells were plated in wells of 2.5cm2 (0.5 x 106) in DMEM medium the day before transfection.

Two Ig of each plasmid DNA construct (wtNS3 / 4A and coNS3 / 4A) was transfected into HepG2 cells using the Fugene 6 (Roche) transfection reagent. After transfection, HepG2 cells were incubated for 24-48 hours.

Preparation and analysis of the protein sample

Cell lysates were analyzed by immunoprecipitation followed by SDS-PAGE. In summary, transiently transfected HepG2 cells were lysed in RIPA buffer (0.15M NaCl, 50mM Tris, 1% Triton-X 100, 1% deoxycholate-Na and 1% SDS). Cell lysates were immunoprecipitated with sepharose protein A and the anti-NS3 polyclonal antibody overnight at 4 ° C. The washed sediments were resuspended in SDS sample buffer, heated at 100 ° C for 5 minutes before SDS-PAGE analysis in 4-12% Bis-Tris gel (Invitrogen) and electrotransferred to nitrocellulose membranes.

NS3 protein expression analysis

The detection of the NS3 protein was performed according to the manufacturer's protocol using a chemiluminescence-bound western-type transfer kit (WesternBreeze; Invitrogen). NS3 protein expression was detected and quantified as a chemiluminescent signal using a NS3 specific polyclonal antibody. Chemiluminescent signals were detected using the GeneGnome (Syngene, Cambridge, United Kingdom). The quantification of Western blots with chemiluminescence was performed in GeneGnome and the intensity units of each protein band were calculated and compared with a standard rNS3 curve.

Semliki forest virus (SFV) vectors

Hamster neonate (BHK) -21 kidney cells were maintained in complete BHK medium supplemented with 5% FCS, 10% tryptose phosphate broth, 2mM glutamine, 20mM HEPES or antibiotics (10 Ig / ml streptomycin and 100 IU / ml of penicillin).

The wtNS3 / 4A gene was isolated by PCR as a Spe1-BStB1 fragment and inserted into the Spe1-BstB1 site of pSFV10Enh containing a translation enhancer sequence of 34 amino acids in length of the capsid, followed by FMDV 2a cleavage peptide . The packaging of the recombinant RNA into rSFV particles was performed using a two-collaborator RNA system. Indirect immunofluorescence of infected BHK cells was performed by determining the titer of recombinant virus stores.

Immunofluorescence

BHK cells were transfected transiently with coNS3 / 4A-pVAX1 according to conventional techniques using lipofectamine plus reagent (Invitrogen) or infected with rSFV. The NS3 protein was detected by indirect immunofluorescence.

Immunization protocols

Groups (5-10 mice / group of BALB / c (H-2d) or C57BL / 6 (H-2b) female mice, 4-8 weeks old, were immunized by injections with 100 Ig water of the plasmid DNA that encodes single or multiple HCV proteins Plasmid DNA in PBS was administered intramuscularly (im) in the anterior tibial muscle (TA). Where indicated in the text, mice im 50 Il / TA of cardiotoxin were injected 0 , 01 mM (Latoxan, Rosans, France) in sterile 0.9% NaCl five days before DNA immunization.The mice received booster doses at four week intervals.

For gene gun banding immunizations, the plasmid DNA was bound to gold particles (1Im) according to the protocols supplied by the manufacturer ((Bio-Rad Laboratories, Hercules, CA). Before immunization, the Abdominal injection area and immunization was performed according to the manufacturer's protocol at a helium discharge pressure of 500 psi. Each injection dose contained 4 Ig of plasmid DNA.The mice received the same booster dose at monthly intervals .

For immunizations with rSFV particles, mice were immunized subcutaneously at the base of the tail with 1 x 107 virus particles diluted in PBS (wtNS3 / 4A-SFV) in a final volume of 100 Il. Peptide immunization was performed by subcutaneous immunization at the base of the tail with 100 Ig peptide mixed at 1: 1 in Freund's complete adjuvant.

ELISA for the detection of murine HCV anti-NS3 antibodies

Serum was collected for the detection of antibodies and isotyping every second or fourth week after the first immunization by retroorbital extraction of mice anesthetized with isofluorane. Enzyme immunoassays were performed as described above.

Cell lines

The myeloma cell line SP2 / 0-Ag14 (H-2d) was maintained in DMEM medium supplemented with 10% fetal bovine serum (FCS, Sigma Chemicals, St. Louis, MO), 2 mM L-Glutamine, 10 mM HEPES , 100 U / ml of penicillin and 100 Ig / ml of streptomycin, 1mM of non-essential amino acids, 1-mercaptoethanol 50 IM, 1mM sodium pyruvate (GIBCO-BRL, Gaithesburgh, MD). SP2 / 0-Ag14 cells with stable NS3 / 4A expression were maintained in 800 Ig of geneticin (G418) / ml of complete DMEM medium.

EL-4 (H-2b) lymphoma cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 10mM HEPES, 1mM sodium pyruvate, 1mM non-essential amino acids, 50m 1-mercaptoethanol, 100 U / ml penicillin and 100 Ig / ml streptomycin (GIBCO-BRL). EL-4 cells with stable expression of NS3 / 4A were generated by transfection of EL-4 cells with the linearized NS3 / 4A-pcDNA3.1 plasmid using the SuperFect transfection reagent (Qiagen GmbH, Hilden, FRG). The transfection procedure was performed according to the manufacturer's protocol. Transfected cells were cloned by limit dilution and selected by the addition of 800 Ig of geneticin (G418) / ml of complete RPMI 1640 medium.

RMA-S cells (a kind gift from Professor Klas Kärre, Karolinska Institutet, Sweden) were kept in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine, 100 U / ml penicillin and 100 Ig / ml of streptomycin. All cells were grown in a humidified incubator at 37 ° C and 5% CO2.

In vivo depletion of T cells

Subpopulations of CD4 and CD8 T cells were depleted in vivo by intraperitoneal injection of purified hybridoma supernatant. A total of 0.4 mg per mouse per injection of anti-CD4 (clone GK1.5) or anti-CD8 (clone 53-6.7) was injected on days -3, -2 and -1 before exposure to the tumor and on days 3, 6, 10, and 13 after the exhibition. Flow cytometric analysis of peripheral blood mononuclear cell populations on days 0, 3, 6, 10 and 13 showed that more than 85% of CD4 and CD8 T cells had been depleted.

In vivo exposure to tumor cells expressing NS3 / 4A

In vivo exposure of mice immunized with the myeloma cell line SP2 / 0 expressing NS3 / 4A or EL-4 lymphoma was performed according to the procedure described by Encke et al., Ant. In summary, groups of BALB / c or C57BL / 6 mice were immunized with different immunogens on weeks zero, four and eight as described. Two weeks after the last immunization, 1 x 106 SP2 / 0 cells were injected subcutaneously

or EL-4 expressing NS3 / 4A on the right flank. The kinetics of tumor growth was determined by measuring the size of the tumor through the skin on days six to 20. The kinetic development of the tumor in two groups of mice was compared using the area under the curve (AUC). Mean tumor sizes were compared using analysis of variance (ANOVA). On day 20 all mice were sacrificed.

Studying the therapeutic effect of the vaccines, tumor cells were inoculated in groups of mice as described above. After six or 12 days the mice were immunized once. Tumor growth was monitored from day 6 to day 20.

Antibodies and MHC fusion protein: Ig

All monoclonal antibodies and MHC: Ig fusion proteins were purchased from BDB Pharmingen (San Diego, CA); Anti-CD16 / CD32 (Fc-block ™, clone 2.4G2), anti-CD8 conjugated with FITC (clone 53-6.7), anti-CD4 conjugated with Cy-Chrome (clone RM4-5), anti-H-2Db conjugate with FITC (clone KH95), H-2Db: recombinant soluble dimeric mouse Ig, rat-a mouse IgG1 conjugated to PE (clone X56).

Detection of specific CTL activity of NS3 / 4A

The splenocytes of C57BL / 6 mice immunized with DNA or sSFV were resuspended in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-Glutamine, 10 mM HEPES, 100 U / ml penicillin and 100 Ig / ml streptomycin, 1mM of non-essential amino acids, 1-mercaptoethanol 50 IM, 1mM sodium pyruvate. In vitro stimulation was carried out for five days in 25 ml flasks at a final volume of 12 ml, containing 5 U / ml of recombinant IL-2murin (mIL-2; R&D Systems, Minneapolis, MN, USA). .). The re-stipulation culture contained a total of 25 x 106 immune splenocytes and 2.5 x 106 irradiated syngeneic EL-4 cells (10,000 rad) expressing the NS3 / 4A proteins. After five days, in vitro stimulation was performed by conventional 51 Cr release assay. Effector cells were collected and a four hour 51 Cr test was performed on 96-well U-bottom plates in a total volume of 200 Il. A total of 1 x 106 target cells (EL-4 cells expressing NS3 / 4A) were marked for one hour at + 37 ° C with 2 Il of 51 Cr (5 mCi / ml) and then washed three times in PBS. To the wells several numbers of 51Cr-labeled effector and target cells (5 x 103 cells / well) were added in effector ratios: target (E: D) of 60: 1, 1 and 7: 1. The level of cytolytic activity was determined after the incubation of the effectors and targets for 4 hours at +37 ° C, 100 Il of the supernatant was collected and the radioactivity was measured with a y-counter.

Splenocytes from mice immunized with DNA or rSFV were collected from C57BL / 6 mice and resuspended in complete RPMI 1640 medium as previously described. In summary, in vitro stimulation was carried out for five days by mixing 25 x 106 splenocytes and 25 x 106 irradiated syngeneic splenocytes (2,000 rad). The re-stipulation was performed in the presence of the NS3 / 4A H-2Db 0.05 IM binding peptide (GAVQNEVTL sequence SEQ ID NO. After the re-stipulation, a four-hour 51 Cr release test was performed using RMA-S cells that received pulses of 51 Cr labeled peptides as targets Cytotoxic activity was determined at proportions of E: D 60: 1, 20: 1 and 7: 1.

The results were expressed according to the formula: percentage of specific lysis = (experimental release - spontaneous release) / (maximum release - spontaneous release). The experimental release is the average / minute count released by the target cells in the presence of the effector cells. Maximum release is the radioactivity released after lysis of the target cells with 10% Triton X-100. Spontaneous release is the leakage of radioactivity into the medium of the target cells.

In Vitro T cell depletion experiments were performed by incubating the effector cells with a hybridoma supernatant containing anti-CD8 monoclonal antibodies (clone RL 172.4; anti-CD4, or clone 31M; anti-CD8) for 30 minutes at 4 ° C. The cells were then washed and incubated at 37 ° C for 1 hour with complement (1/20 dilution of low toxic rabbit complement, Saxon, UK) before performing the CTL assay described above.

Quantification of specific CT3 of NS3 / 4A by flow cytometry

The frequency of specific CD8 + T cells of the NS3 peptide was analyzed by ex-vivo staining of splenocytes from mice immunized with DNA or rSFV with the H-2Db: recombinant soluble dimeric mouse Ig fusion protein as described above. In summary, splenocytes were resuspended in PBS / 1% FCS (buffer for FACS) and incubated with Fc blocking antibodies. Then, the cells were washed and incubated with H2Db: Ig preloaded with NS3 / 4th derivative peptide. The cells were then washed and incubated with rat-a mouse IgG1 antibody conjugated to PE, mouse CD8 antibody conjugated to FITC and mouse CD4 antibody to Cy-Chrome. After washing, the cells were diluted in FACS buffer containing propidium iodide (PI). Approximately 200,000 total events from each sample were acquired in a FACS Calibur (BDB) and dead cells (PI-positive cells) were excluded from the analysis.

Statistic analysis

Fisher's exact test was used analyzing the frequency and the Mann-Whitney U test was used comparing values from two groups. Kinetic tumor development in two groups of mice was compared using the area under the curve (AUC). The AUC values were compared using the analysis of variance (ANOVA). The calculations were performed using the Macintosh version of the StatView software (version 5.0).

Some of the peptide embodiments of the invention are described in the following section.

HCV peptides

HCV peptides encompassed or derived therefrom include, but are not limited to, those containing as primary amino acid sequence the entire amino acid sequence as represented in the sequence listing (SEQ ID NO: 2-11 and SEQ ID NO: 36) and fragments of SEQ ID. No. 2-11 and SEQ ID No. 36 having a length of at least four amino acids (e.g., SEQ ID NO: 14-16) including altered sequences in which functionally equivalent amino acid residues are substituted by residues within the sequence that result in a silent change. Preferred fragments of a sequence of SEQ ID. No. 2-11 and SEQ ID No. 36 have a length of at least four amino acids and comprise a unique amino acid sequence for the discovered NS3 / 4A peptide or mutants thereof, including altered sequences in which the functionally equivalent amino acid residues are replaced by residues within the sequence that result in a silent change. VCH peptides may have a length of, for example, at least 12-704 amino acids (e.g., a length of any number between 12-15, 15-20, 20-25, 25-50, 50-100 , 100-150, 150-250, 250-500 or 500-704 amino acids).

Embodiments also include HCV peptides that are substantially identical to those described above. That is, HCV peptides that have one or more amino acid residues in SEQ ID NO: 2-11 and SEQ ID NO: 36 and fragments thereof that are substituted by another amino acid of a similar polarity, which acts as a functional equivalent, which results in a silent alteration. In addition, HCV peptides that have one or more amino acid residues in SEQ ID NO. 2-11 and SEQ ID NO. 36 or a fragment thereof provided that the fusion does not significantly alter the structure or function (eg. , immunogenic properties) of the HCV peptide. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. Examples of aromatic amino acids include phenylalanine, tyrosine and tryptophan. Accordingly, it is said that the peptide embodiments of the invention consist essentially of SEQ ID NOS 2-27 and SEQ ID N ° 36 in the light of the modifications described above.

The HCV peptides described herein can be prepared by chemical synthesis procedures (such as solid phase peptide synthesis) using techniques known in the art, such as those set forth by Merrifield et al., J. Am. Chem. Soc 85: 2149 (1964), Houghten et al., Proc. Natl Acad. Sci. USA,

82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis, Pierce Chem Co., Rockford, IL (1984) and Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY These polypeptides they can be synthesized with or without a methionine at the amino terminus. Chemically synthesized HCV peptides can be oxidized using procedures set forth in these references to form disulfide bridges.

Although the HCV peptides described herein can be chemically synthesized, it may be more effective to produce these polypeptides by recombinant DNA technology. Such procedures can be used by constructing expression vectors containing the HCV nucleotide sequences described above and suitable transcription and translation control signals. These procedures include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Alternatively, RNA capable of encoding a VHX nucleotide sequence can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford. Accordingly, several cell lines that agree to the embodiments have been engineered to express the encompassed VCH peptides. For example, some cells are created by expressing the VCH peptides of SEQ ID NO: 2-11 and SEQ ID NO: 36 or fragments of these molecules (eg, SEQ ID NO: 14-26).

By expressing the encompassed HCV peptides several host-expression vector systems can be used. Suitable expression systems include, among others, microorganisms such as bacteria (eg, E. coli or B. subtilis) transformed with recombinant bacteriophage, plasmid DNA or cosmid DNA expression vectors, containing sequences of antibody coding; yeasts (Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing HCV nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) containing HCV sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing HCV sequences; or mammalian cell systems (eg, COS, CHO, BHK, 293, NS0 and 3T3 cells) that house recombinant expression constructs containing promoters derived from the mammalian cell genome (eg, metallothionein promoter ) or of mammalian viruses (eg, the adenovirus late promoter, the 7.5 K promoter of vaccinia virus).

In bacterial systems, advantageously, numerous expression vectors can be selected based on the intended use for the HCV gene product that is expressed. For example, when a large amount of said protein is to be produced, for the generation of pharmaceutical compositions of the HCV peptide or producing antibodies against the HCV peptide, for example, vectors that direct the expression of high levels of fusion protein products They are easily purified. Such vectors include, among others, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 2: 1791), in which the HCV coding sequence can be individually linked to the vector within the framework of the lacZ coding region, so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13: 3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem., 264: 5503-5509 (1989)); and the like PGEX vectors can also be used expressing foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can be purified from lysed cells by adsorption on glutathione-agarose beads, followed by elution in the presence of free glutathione. PGEX vectors have been designed to include thrombin or cleavage sites by factor Xa protease so that the cloned target gene product can be released from the GST fraction.

In an insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The HCV coding sequence can be cloned individually in non-essential regions (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter, for example the polyhedrin promoter. Successful insertion of an HCV gene coding sequence will result in inactivation of the polyhedrin gene and the production of non-occluded recombinant viruses (i.e., viruses that lack the proteinaceous coating encoded by the polyhedrin gene). These recombinant viruses are then used to infect S. frugiperda cells in which the inserted gene is expressed. (See, for example, Smith et al., J. Virol. 46: 584 (1983); and Smith, U.S. Patent No. 4,215,051).

In mammalian host cells, numerous virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the HCV nucleotide sequence of interest can be linked to a transcription / translation control adenovirus complex, for example the late promoter and the tripartite leader sequence. Then, this chimeric gene can be inserted into the adenovirus genome by recombination in vitro or in vivo. Insertion into a non-essential region of the viral genome (eg, E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the HCV gene product in infected hosts. (See, for example, Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 3655-3659 (1984)). Specific initiation signals may also be required for the effective translation of the HCV nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences.

However, in cases where only a portion of the HCV coding sequence is inserted, exogenous translational control signals may be provided including, perhaps, the ATG initiation codon. In addition, the initiation codon has to be in phase with the reading frame of the desired coding sequence guaranteeing the translation of the entire insert. These exogenous translation control signals and initiation codons can have diverse origins, both natural and synthetic. Expression efficiency can be enhanced by the inclusion of suitable transcription enhancer elements, transcription terminators etc. (see, Bittner et al., Methods in Enzymol., 153: 516-544 (1987)).

In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences or that modifies and processes the gene product in the specific way desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for the processing and post-translational modification of proteins and gene products. Suitable cell lines or host systems can be chosen so as to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery for the proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, among others, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38.

Stable expression is preferred for long-term production and high yield of recombinant proteins. For example, cell lines that stably express the HCV peptides described above can be engineered. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by suitable expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, sites of polyadenylation etc.) and a selectable marker. After the introduction of the foreign DNA, the engineered cells are allowed to grow for 1-2 days in an enriched medium and then passed to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow into foci that, in turn, clone and expand in cell lines. This procedure is advantageously used by subjecting the cell lines that express the HCV gene product to engineering.

Numerous selection systems can be used, including, among others, the thymidine kinase genes of the herpes simplex virus (Wigler et al., 1977, Cell 11: 223), glutamine synthetase, hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 2026 (1962)) and the adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817-17 (1980)) genes can be used in tk-, gs cells -, hgprt- or aprt, respectively. Likewise, antimetabolite resistance can be used as the basis of the selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 30, Proc. Natl. Acad. Sci. USA, 77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA, 78: 2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981)); and higro, which confers hygromycin resistance (Santerre et al., Gene 30: 147 (1984)).

Alternatively, any fusion protein can be easily purified using a specific antibody of the fusion protein being expressed. For example, a system described by Janknecht et al. It allows easy purification of non-denatured fusion proteins expressed in human cell lines. (Janknecht et al., Proc. Natl. Acad. Sci. USA 88: 8972-8976 (1991)). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid so that the gene's open reading frame is condensed translationally with a marker on the terminal amino consisting of six histidine residues. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2 + agarose-nitrileacetic acid columns and histidine-labeled proteins are selectively eluted with imidazole-containing buffers. The following example describes a procedure that was used expressing HCV peptides encoded by the encompassed nucleic acids.

Example 8

Characterizing the NS3 / 4A-pVAX, MSLF1-pVAX and NS3 / 4A mutant constructs described in Example 1, the plasmids were transcribed and translated in vitro and the resulting polypeptides were visualized by sodium polyacrylamide dodecyl sulfate gel electrophoresis. In vitro transcription and translation were performed using the T7-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions. All in vitro translation reactions of the expression constructs were carried out at 30 ° C with 35S methionine (Amersham International, P1c, Buckinghamshire, United Kingdom). The labeled proteins were separated by 12% SDS-PAGE and visualized by exposure to X-ray film (Hyper Film-MP, Amersham) for 6-18 hours.

In Vitro analysis revealed that all proteins were expressed in high amounts of their respective expression constructs. The rNS3 construct (NS3-pVAX vector) produced a single peptide of approximately 61 kDa, while the mutant constructs (e.g., the TGT construct (NS3 / 4A-TGTpVAX) and the RGT construct (NS3 / 4A-RGT -pVAX)) produced a single polypeptide of approximately 67 kDa, which is identical to the molecular weight of the uncleaved NS3 / 4A peptide produced from the NS3 / 4ApVAX construct. The cleaved product produced from the expressed NS3 / 4A peptide was approximately 61 kDa, which was identical in size to rNS3 produced from the NS3-pVAX vector. These results demonstrated that the expression constructs were functional, the NS3 / 4A construct was enzymatically active, rNS3 produced a peptide of the predicted size and the cut-off mutations completely abolished the cleavage at the NS3-NS4A junction.

Comparing the translation efficiency of the NS3 / 4A-pVAX and MSLF1-pVAX plasmids, the amount of input DNA was serially diluted before addition to the assay. Serial dilutions of the plasmids revealed that the MSLF1 plasmid gave stronger bands at higher dilutions of the plasmid than the wild-type NS3 / 4A plasmid, providing indications that transcription and translation in vitro was more efficient from plasmids. MSLF1. Plasmids NS3 / 4A-pVAX and MSLF1 were then analyzed for protein expression using transiently transfected Hep-G2 cells. Similar results were obtained in that the MSLF1 gene provided a more efficient expression of NS3 than the native NS3 / 4A gene.

The sequences, constructs, vectors, clones and other materials comprising the VCH nucleic acids of the embodiment and the peptides may be in enriched or isolated form. As used herein

document, "enriched" means that the concentration of the material is many times its natural concentration, for

example at least about 5, 10, 100, or 1000 times its natural concentration, advantageously 0.01% by weight, preferably at least about 0.1% by weight. Enriched preparations of approximately 0.5% or more are also contemplated, for example%, 5%, 10% and 20% by weight. The term "isolated" requires that the material be removed from its original environment (eg, the natural environment if it is of natural origin). For example, a natural polynucleotide present in the natural state in a living animal is not isolated, although the same polynucleotide separated from some or all of the coexisting materials in the natural system is considered

isolated. It is also advantageous that the sequences may be in purified form. The term "purified" does not

it requires absolute purity; instead, it is intended to be a relative definition. The isolated proteins have been conventionally purified to electrophoretic homogeneity by, for example, Coomassie staining. The purification of the starting material or the natural material up to at least an order of magnitude, preferably two

or three orders and more preferably, four or five orders of magnitude is expressly contemplated.

The HCV gene products described herein can also be expressed in plants, insects and animals so that a transgenic organism is created. Desirable transgenic plant systems that have an HCV peptide include Arabadopsis, corn and Chlamydomonas. Desirable insect systems that have an HCV peptide include, among others, D. melanogaster and C. elegans. Animals of any species, including, among others, amphibians, reptiles, birds, mice, hamsters, rats, rabbits, guinea pigs, pigs, dwarf pigs, goats, dogs, cats and non-human primates, for example baboons, monkeys and chimpanzees, are they can use generating transgenic animals that have an encompassed HCV molecule. These transgenic organisms desirably exhibit the transfer to the germ line of the HCV peptides described herein.

Preferably, any technique known in the art is used by introducing HCV transgenes into animals producing the founding lines of transgenic animals or eliminating or replacing existing HCV genes. Such techniques include, among others, pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Patent No. 4,873,191); gene transfer mediated by retrovirus in germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)); gene direction in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)); embryo electroporation (Lo, Mol Cell. Biol. 3: 1803-1814 (1983); and sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989)); see also Gordon, Transgenic Animals, Intl. Rev. Cytol. 115: 171-229 (1989).

After synthesis or expression and isolation or purification of HCV peptides, the isolated or purified peptide can be used to generate antibodies. Depending on the context, the term "antibodies" may encompass antibodies, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that recognize HCV peptides have many uses, including but not limited to biotechnological applications, therapeutic / prophylactic applications and diagnostic applications.

For the production of antibodies, several hosts, including goats, rabbits, rats, mice and humans etc., can be immunized by injection with an HCV peptide. Depending on the host species, several adjuvants can be used to increase the immune response. Such adjuvants include ribavirin, Freund's adjuvants, mineral gels such as aluminum hydroxide and active surface substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, Californian barnacle hemocyanin and dinitrophenol. BCG (Calmette-Guerin bacillus) and Corynebacterium parvum are also potentially useful adjuvants.

The peptides used to induce specific antibodies may have an amino acid sequence consisting of at least four amino acids and preferably, at least 10 to 15 amino acids. By one approach, short stretches of amino acids encoding NS3 / 4A fragments are condensed with those of another protein, such as Californian limpet hemocyanin, in such a way that an antibody against the chimeric molecule is produced. Additionally, a composition comprising ribavirin and an HCV peptide (SEQ ID No. 2-11 and SEQ ID No. 36), a fragment thereof containing any number of consecutive amino acids between at least 3-50 amino acids (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive amino acids) (e.g., SEQ ID NO: 4-26), or a nucleic acid that encoding one or more of these molecules is administered to an animal, preferably a mammal, including a human being. Although antibodies capable of specifically recognizing HCV can be generated by injecting synthetic peptides of 3 units, 10 units and 15 units corresponding to an HCV peptide into mice, a more diverse set of antibodies can be generated using HCV peptides recombinants, prepared as described above.

By generating antibodies against an HCV peptide, a substantially pure peptide is isolated from a transfected or transformed cell. The concentration of the peptide in the final preparation is adjusted by, for example, concentration of an Amicon filter device, at the level of a few microorganisms / ml. The monoclonal or polyclonal antibody against the peptide of interest can be prepared as follows:

Monoclonal antibodies against a VCH peptide can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, among others, the hybridoma technique initially described Koehler and Milstein (Nature 256: 495-497 (1975)), the human B cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983)); Cote et al Proc Natl Acad Sci 80: 2026-2030 (1983) and the EBV hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pages 77-96 (1985). In addition, developed techniques can be used

for the production of "chimeric antibodies", the splicing of mouse antibody genes to genes of

human antibodies obtaining a molecule with the appropriate antigenic specificity and biological activity. (Morrison et al. Proc Natl Acad Sci 81: 6851-6855 (1984); Neuberger et al. Nature 312: 604-608 (1984); Takeda et al. Nature 314: 452-454 (1985)). Alternatively, the techniques described for the production of single chain antibodies (US Patent 4,946,778) can be adapted by producing HCV specific single chain antibodies. Antibodies can also be produced by induction of in vivo production of the lymphocyte population or by selective detection of highly specific immunoglobulin libraries or panels of binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833 -3837 (1989) and Winter G. and Milstein C; Nature 349: 293-299 (1991).

Antibody fragments containing specific binding sites of an HCV peptide can also be generated. For example, said fragments include, among others, the F (ab ') 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fav fragments that can be generated by reducing the disulfide bridges of the F fragments. (ab ') 2. Alternatively, Fab expression libraries can be constructed allowing quick and easy identification of monoclonal Fb fragments with the appropriate specificity. (Huse W. D. et al. Science 256: 1275-1281 (1989)).

By one approach, monoclonal antibodies against an HCV peptide are manufactured as follows. Briefly, a few micrograms of the selected protein or peptides derived from it are repeatedly inoculated over a period of a few weeks. The animal is then sacrificed and the antibody producing cells are isolated from the spleen. Splenocytes are condensed in the presence of polyethylene glycol with mouse myeloma cells and excess uncondensed cells are destroyed by the growth of the system in selective medium comprising aminopterin (HAT medium). Successfully diluted condensed cells and aliquots of the dilution are introduced into wells of a microtiter plate in which culture growth is continued. Antibody producing clones are identified by detection of antibodies in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as initially described by Engvall, E., Meth. Enzymol 70: 419 (1980) and procedures derived therefrom. Selected positive clones can be expanded and the monoclonal antibody product can be collected for use. Detailed procedures for the production of monoclonal antibodies are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

The polyclonal antiserum containing antibodies against heterogeneous epitopes of a single protein can be prepared by immunization of suitable animals with the expressed protein or peptides thereof described above, which may be unmodified or modified by enhancing immunogenicity. The effective production of polyclonal antibodies is affected by many factors related to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of vehicles and adjuvants. Likewise, host animals vary with respect to the response to the site of inoculations and the dose, with both inadequate and excessive doses of the antigen resulting in low titer antisera. Small doses (ng levels) of the antigen administered at multiple intradermal sites appear to be the most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrine / .Metab. 33: 988-991 (1971).

Booster injections are provided at regular intervals and the antiserum is collected when the antibody titre thereof, semiquantitatively determined, for example by double agar immunodiffusion against known concentrations of the antigen, begins to decrease. See, for example, Ouchterlony, O. et al., Chapter 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). The equilibrium concentration of the antibody is usually in the range of 0.1 to 0.2 mg / ml of serum (approximately 12 IM). The affinity of the antiserum for the antigen is determined by preparing competitive binding curves, as described in, for example, Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2nd Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibody preparations prepared according to the protocol are useful in quantitative immunoassays that determine concentrations of antibody carrying substances in biological samples; they are also used semiquantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of HCV in biological samples). The following section describes how some of the nucleic acids and peptides described above can be used in diagnostics.

Diagnostic realizations

In general, the covered diagnoses are classified according to whether a nucleic acid or protein based assay is used. Some diagnostic tests detect the presence or absence of a VCH nucleic acid sequence encompassed in a sample obtained from a patient, while other assays seek to identify whether an encompassed VCH peptide is present in a biological sample obtained from a patient. Additionally, the manufacture of kits incorporating the reagents and procedures described herein that allow rapid detection and identification of HCV is also encompassed. These diagnostic kits may include, for example, a nucleic acid probe or antibody that specifically detects HCV. The detection component of these kits will normally be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding DNA, RNA or proteins will often be provided. Available supports include nitrocellulose, nylon or derived nylon membranes that can be characterized by carrying a matrix of positively charged substituents. One or more restriction enzymes, control reagents, buffers, amplification enzymes and non-human polynucleotides such as salmon sperm or calf thymus DNA can be supplied in these kits.

Useful diagnoses based on nucleic acid include, but are not limited to, direct DNA sequencing, Southern type transfer analysis, point transfer analysis, nucleic acid amplification and combinations of these approaches. The starting point for these analyzes is isolated or purified nucleic acid from a biological sample obtained from a patient suspected of having contracted HCV or a patient at risk of contracting HCV. The nucleic acid extracted from the t-sample can be amplified by RT-PCR and / or DNA amplification using primers corresponding to regions flanking the HCV nucleic acid sequences encompassed.

(e.g., NS3 / 4A (SEQ ID NO. 1)).

In some embodiments, the nucleic acid probes that specifically hybridize with HCV sequences are attached to a support in an ordered matrix, in which the nucleic acid probes are fixed to different regions of the support that do not overlap each other. Preferably, said array is designed to be

“Approachable” when the different locations of the probe are registered and can be accessed as part of a

test procedure These probes are attached to a support in different known locations. He

knowledge of the precise location of each nucleic acid probe makes these matrices "treatable"

particularly useful in binding assays. The nucleic acids of a preparation of several biological samples are then labeled by conventional approaches (eg, radioactivity or fluorescence) and the labeled samples are applied to the matrix under conditions that allow hybridization.

If a nucleic acid in the samples hybridizes with a probe in the matrix, a signal will be detected at a position of the support corresponding to the location of the hybrid. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied, an identification of the presence of the polymorphic variant can be determined quickly. These approaches are easily automated using technology known to those skilled in the art of high performance detection or diagnostic analysis.

Additionally, an opposite approach to the one presented above can be used. Nucleic acids present in biological samples can be arranged on a support creating an approachable matrix. Preferably, the samples are placed on the support in known positions that do not overlap. The presence of VCH nucleic acids in each sample is determined by applying labeled nucleic acid probes that complement the nucleic acids, which encode VCH peptides, at locations on the matrix corresponding to the positions in which the biological samples were placed. Since the identity of the biological sample and its position on the matrix is known, the identification of a patient who has been infected with HCV can be quickly determined. These approaches are also easily automated using technology known to those skilled in the art of high performance diagnostic analysis.

Any approachable matrix technology known in the art can be used. A concrete realization of

Polynucleotide matrices is known as Genechips ™ and has generally been described in US Pat.

5,143,854; PCR publications WO 90/15070 and 92/10092. These nuances are generally produced using mechanical synthesis procedures or light-directed synthesis procedures, which incorporate a combination of photolithographic procedures and solid phase oligonucleotide synthesis. (Fodor et al., Science, 251: 767-777, (1991)). The immobilization of oligonucleotide matrices on solid supports has been made possible by the

development of a technology generally identified as "Immobilized polymer synthesis on a very large scale" (VLSPIS ™) in which, normally, the probes are immobilized in a high density matrix on a solid surface on a chip. Examples of VLSPIS ™ technologies are provided in US Pat. 143,854 and

5,412,087 and PCR publications WO 90/15070, WO 92/10092 and WO 95/11995, which describe methods forming oligonucleotide matrices by techniques such as light-directed synthesis techniques. In the design of strategies aimed at providing oligonucleotide matrices immobilized on solid supports, other presentation strategies were developed by ordering and displaying oligonucleotide matrices on the chips in an attempt to maximize hybridization patterns and diagnostic information. Examples of these presentation strategies are disclosed in publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256 ..

Those skilled in the art know a wide variety of labels and conjugation techniques and can be used in various nucleic acid assays. There are several ways to produce labeled nucleic acids for hybridization or PCR, including, but not limited to, oligomarking, translation in traveling dent, terminal labeling or PCR amplification using a labeled nucleotide. Alternatively, a nucleic acid encoding HCV can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art and are commercially available and can be used by synthesizing RNA probes in vitro by the addition of a suitable RNA polymerase, such as T7, T3 or SP6 and labeled nucleotides. A number of companies, such as Pharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.) And U.S. Biochemical Corp (Cleveland Ohio) supply commercial kits and protocols for these procedures. Suitable indicator molecules or markers include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like.

The presence of an HCV peptide in a protein sample obtained from a patient can also be detected using conventional assays and the embodiments described herein. For example, antibodies that are immunoreactive with the HCV peptides disclosed by screening biological samples can be used to determine the presence of HCV infection. In preferred embodiments, antibodies that are reactive for the encompassed HCV peptides are used by immunoprecipitating the HCV peptides disclosed from biological samples or used by reacting them with proteins obtained from a biological sample in western blots or immunoblotting. Favored diagnostic embodiments also include enzyme-linked immunosorption assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEAM), including sandwich assays using specific monoclonal and / or polyclonal antibodies to HCV peptides disclosed. Examples of sandwich tests are described in David et al., In US Pat. No. 4,376,110 and 4,486,530. Other embodiments employ aspects of the immune strip technology disclosed in US Pat. No. 5,290,678; 5,604,105; 5,710,008; 5,744,358; and 5,747,274.

In another preferred protein-based diagnosis, the antibodies described herein are fixed to a support in an ordered matrix, in which a plurality of antibodies are fixed to different regions of the support that do not overlap each other. As with nucleic acid-based matrices, protein-based matrices are ordered matrices that are designed to be "approachable" so that different locations are registered and can be accessed as part of an assay procedure. These probes are attached to a support in different known locations. The knowledge of the precise location of each probe makes

these "approachable" matrices particularly useful in binding assays. For example, an approachable matrix can

comprising a support having several regions to which a plurality of antibody probes are attached that specifically recognize HCV peptides present in a biological sample and differentiate the HCV isotype identified herein.

By one approach, proteins are obtained from biological samples and then labeled by conventional approaches (eg, radioactivity, colorimetrically or by fluorescence). Then, the labeled samples are applied to the matrix under conditions that allow binding. If a protein in the sample binds to an antibody probe on the matrix, a signal will be detected at a position of the support corresponding to the location of the antibody-protein complex. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied, an identification of the presence, concentration and / or level of expression can be determined rapidly. That is, using labeled standards of a known concentration of the HCV peptide, a researcher can accurately determine the protein concentration of the particular peptide in an analyzed sample and can also assess the level of HCV peptide expression. Conventional procedures can also be used by determining more precisely the concentration or level of HCV peptide expression. These approaches are easily automated using technology known to those skilled in the art of high performance diagnostic analysis.

In another embodiment, an opposite approach to the one presented above can be used. Nucleic proteins present in biological samples can be arranged on a support creating an approachable matrix. Preferably, the protein samples are placed on the support in known positions that do not overlap. The presence of an HCV peptide in each sample is determined by applying labeled antibody probes that recognize specific epitopes of the HCV peptide. Since the identity of the biological sample and its position on the matrix is known, an identification of the presence, concentration and / or level of expression of an HCV peptide can be determined rapidly.

That is, using labeled standards of a known concentration of the HCV peptide, a researcher can accurately determine the concentration of the peptide in a sample and from this information, can assess the level of expression of the peptide. Conventional procedures can also be used by determining more precisely the concentration or level of HCV peptide expression. These approaches are also easily automated using technology known to those skilled in the art of high performance diagnostic analysis. As detailed above, any approachable matrix technology known in the art can be used. The following section describes more compositions that include the HCV nucleic acids and / or HCV peptides described herein.

Compositions comprising HCV nucleic acids or peptides

Embodiments of the invention also include NS3 / 4A fusion proteins or nucleic acids encoding these molecules. For example, the production and purification of recombinant protein can be facilitated by the addition of auxiliary amino acids forming a "tag." Such tags include, among others, His-6, Flag, Myc and GST. The tags can be added to the C-terminus, the N-terminus or within the amino acid sequence of NS3 / 4th. Other embodiments include NS3 / 4A fusion proteins with truncations at the amino or carboxy ends, or internal deletions, or with additional polypeptide sequences added to the amino or carboxy ends or added internally. Other embodiments include NS3 / 4A fusion proteins or truncated or mutated versions thereof, in which the residues of the proteolytic cleavage site of NS3 / 4th have been replaced. Such substitutions include, among others, sequences in which the P1 'site is a Ser, Gly, or Pro, or the P1 position is an Arg, or in which the sequence P8 to P4' is Ser-Ala-Asp-Leu -Glu-Val-Val-Thr-Ser-Thr-Trp-Val (SEQ ID No. 15).

More embodiments relate to an immunogen comprising the NS3 / 4A fusion protein or a truncated, mutated or modified version thereof, capable of producing an enhanced immune response against NS3. The immunogen can be provided in a substantially purified form, which means that the immunogen substantially lacks other proteins, lipids, carbohydrates or other compounds with which it is naturally associated.

Some embodiments contain at least one of the HCV nucleic acids or HCV peptides (eg, SEQ ID NOS. 1-27, 35, or 36) attached to a support. Preferably, these supports are manufactured in a manner that creates a multimeric agent. These multimeric agents provide the VCH peptide or nucleic acid in a form such or in a manner that sufficient affinity for the molecule is achieved. A multimeric agent having a VCH nucleic acid or peptide can be obtained by attaching the desired molecule to a macromolecular support. A

"Support" may be referred to as a vehicle, protein, resin, cell membrane, capsid or portion thereof, or

any macromolecular structure joining or immobilizing said molecules. Solid supports include, among others, the well walls of a reaction tray, test tubes, polystyrene spheres, magnetic spheres nitrocellulose strips, membranes, microparticles such as latex particles, animal cells, Duracyte®, artificial cells and others. An HCV nucleic acid or peptide can also be attached to inorganic carriers, such as silicon oxide material (e.g., silica gel, zeolite, diatomaceous earth or aminated glass) by, for example, a covalent bond through of a hydroxy, carboxy or amino group and a reactive group in the vehicle.

In several multimeric agents, the macromolecular support has a hydrophobic surface that interacts with a portion of the VCH nucleic acid or peptide by a non-covalent hydrophobic interaction. In some cases, the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which the hydrophobic groups have joined such as polystyrene, polyethylene or polyvinyl. Additionally, the VCH nucleic acid or peptide can be covalently linked to vehicles, including proteins and oligo / polysaccharides (eg, cellulose, starch, glycogen or aminated sepharose). In these latter multimeric agents a reactive group is used on the molecule, such as a hydroxy or amino group, joining a reactive group on the vehicle creating the covalent bond. Additional multimeric agents comprise a support that has other reactive groups that are chemically activated such that they bind the HCV peptide nucleic acid. For example, cyanogen bromide supports, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chlorformiate and N-hydroxy succinimide chloroformate or acrylic oxirane supports are used. (Sigma).

Vehicles for use in the body (ie, for prophylactic or therapeutic applications) are physiological, non-toxic and preferably, non-immuno-responders. Suitable vehicles for use in the body include poly-L-lysine, poly-D, L-alanine, liposomes, capsids showing the desired HCV peptide or nucleic acid and Chromosorb® (Johns-Manville Products, Denver Co.). Chromosorb® conjugated ligand (Synsorb-Pk) has been analyzed in humans for the prevention of hemolytic uremic syndrome and was reported as having no adverse reactions. (Armstrong et al. J. Infectious Diseases 171: 1042-1045 (1995)). For some embodiments, it

administers a "naked" vehicle (ie lacking a bound HCV nucleic acid or peptide) that has the

ability to bind a nucleic acid or HCV peptide in the body of an organism. Through this approach, a “prodrug-type” therapy has been devised in which the naked vehicle is administered separately from the HCV nucleic acid or peptide and once both are in the body of the organism, the vehicle and the nucleic acid or HCV peptide are mounted in a multimeric complex.

The insertion of linkers (eg, "linkers I" engineered simulating the flexible regions of phage I) of a suitable length between the VCH nucleic acid or peptide and the support is also contemplated by stimulating greater flexibility of the peptide of the HCV, hybrid or binding partner and thus overcome any steric hydration that the support may present. The determination of a suitable length of the linker that allows an optimal cellular response or lack thereof, can be determined by screening the HCV nucleic acid or peptide with several linkers in the tests detailed in the present disclosure.

A composite support comprising more than one type of HCV nucleic acid or peptide is also provided. A "composite support" can be a vehicle, a resin or any macromolecular structure used by joining or immobilizing two or more different HCV nucleic acids or peptides. As before, the insertion of linkers, such as linkers I of a suitable length between the HCV nucleic acid or peptide and the support is also contemplated by stimulating greater flexibility in the molecule and thus, overcoming any steric hydration that may occur. The determination of a suitable length of the linker that allows an optimal cellular response or lack thereof, can be determined by screening the HCV nucleic acid or peptide with several linkers in the tests detailed in the present disclosure.

In other embodiments, the multimeric supports and compounds discussed above may have nucleic acids or multimerized HCV peptides linked so that we create a "multimerized multimeric support" and a "multimerized composite support," respectively. A multimerized ligand can be obtained by, for example, coupling two or more nucleic acids or HCV peptides in tandem using conventional techniques in molecular biology. The multimerized form of the HCV nucleic acid or peptide may be advantageous for many applications by the ability to obtain an agent with a higher affinity, for example. The incorporation of linkers or spacers, such as flexible linkers I, between the individual domains that form the multimerized agent may also be advantageous for some embodiments. The insertion of linkers I of a suitable length between protein binding domains, for example, can stimulate greater flexibility in the molecule and can overcome any steric hydration. Similarly, insertion of linkers between the multimerized HCV nucleic acid or peptide and the support can stimulate greater flexibility and limit the steric hydration presented by the support. The determination of a suitable length of the linker can be performed by screening the HCV nucleic acids or peptides in the tests detailed in the present disclosure.

Embodiments also include vaccine compositions and immunogen preparations comprising the NS3 / 4A fusion protein or a truncated or mutated version thereof and optionally, an adjuvant. The following section describes some of these compositions in greater detail.

Vaccine compositions and immunogenic preparations

Vaccine compositions and immunogenic preparations comprising, consisting of or consisting essentially of HCV nucleic acid or the HCV peptide encompassed or both are contemplated (eg, any one or more of SEQ ID NO: 1-27, 35 or 36). These compositions usually contain an adjuvant, but do not necessarily require an adjuvant. That is, many of the nucleic acids and peptides described herein function as immunogens when administered net. The compositions described herein

(p.

 eg, immunogens and HCV vaccine compositions containing an adjuvant, such as ribavirin) can be manufactured according to conventional procedures of the medical pharmacy producing drugs for administration to animals, for example to mammals, including humans.

Several nucleic acid-based vaccines are known and it is contemplated that these compositions and approaches to immunotherapy can be augmented by reformulation with ribavirin (see, for example, U.S. Patent No. 5,589,466 and 6,235,888). Through an approach, for example, a gene encoding one of the HCV peptides described herein (e.g., SEQ ID No. 1 or SEQ ID No. 35) is cloned into an expression vector capable of expressing the polypeptide. when you enter a subject. The expression construct is introduced into the subject in a mixture of adjuvant (e.g., ribavirin) or together with an adjuvant (e.g., ribavirin). For example, the adjuvant

(p.

 eg, ribavirin) is administered shortly after expression construction in the same place. Alternatively, the subject is provided with the RNA encoding the polypeptide antigen of interest in a mixture with ribavirin or together with an adjuvant (eg, ribavirin).

When the antigen is to be DNA (e.g., prepared from a DNA vaccine composition), suitable promoters include the simian virus 40 promoter (SV4), mouse breast tumor virus (MMTV), virus of human immunodeficiency (HIV), such as the long terminal repeat promoter (LTR) of HIV, Moloney virus, ALV, cytomegalovirus (CMV) such as the early CMV promoter, Epstein Barr virus (EBV), Rous sarcoma virus (RSV) as well as promoters of human genes, such as human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein. Examples of polyadenylation signals useful with some embodiments, especially in the production of a genetic vaccine for humans, include, among others, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal, which is in plasmid pCEP4 (Invitrogen, San Diego Calif.), Called SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for gene expression, other elements can also be included in a gene construct. Such additional elements include enhancers. The enhancer can be selected from the group that includes, among others: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those of CMV, RSV and EBV. Gene constructs can be provided with mammalian replication origin in order to maintain the extrachromosomal construct and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, CA) contain the origin of Epstein Barr virus replication and the EBNA-1 coding region of the nuclear antigen, which produces episomal replication with a high copy count without integration. All forms of DNA can be used, in replication and without being in replication, that are not integrated into the genome and that can be expressed. Preferably, the genetic vaccines comprise ribavirin and a nucleic acid encoding NS3 / 4A NS3, or a fragment or mutant thereof (SEQ ID NO: 2-26 and 36). The following example describes the preparation of a genetic vaccine suitable for use in humans.

Example 9

A VCH expression plasmid is designed expressing the NS3 / 4A peptide (SEQ ID No. 2 or SEQ ID No. 36). The NS3 / 4A coding sequence of NS3 / 4A-pVAX or MSLF1-pVAX is enzymatically removed and the isolated fragment is inserted into plasmid A so that it is subject to transcriptional control of the CMV promoter and the RSV enhancer element. (See, U.S. Patent No. 6,235,888 issued to Pachuk et al.). The structure of plasmid A is 3969 base pairs in length; It contains an origin of PBR replication replicating in E. coli and a kanamycin resistance gene. Inserts such as NS3 / 4A or codon optimized NS3 / 4A are cloned into a polyline region, which places the insert between and operably linked to the promoter and the polyadenylation signal. The transcription of the cloned inserts is subject to the control of the CMV promoter and the RSV enhancer elements. A polyadenylation signal is provided by the

presence of a SV40 polyA signal located just 3 'from the cloning site. Then, one is manufactured

vaccine composition or immunogenic preparation containing NS3 / 4A by mixing any amount of construction between about 0.5-500 mg, for example between 0.5-1 g, 1-2 Ig, 2-5 Ig, 5-10 Ig, 10-20 Ig, 2050I g, 50-75 Ig, 75-100 Ig, 100-250 Ig, 250 Ig-500 Ig with any amount of ribavirin between approximately 0.1-10 mg, for example between 0.1 mg - 0.5 mg, 0.5 mg-1 mg, 1 mg-2 mg, 2 mg-5 mg, or 5 mg-10 mg of ribavirin.

Said vaccine composition can be used to produce antibodies in a mammal (eg, mice or rabbits) or can be injected intramuscularly into a human being producing antibodies, preferably a human being who is chronically infected with the virus of the HCV The recipient preferably receives three immunization boosters of the mixture at 4 week intervals, as well. Through the third booster, the specific HCV antibody titer will increase significantly. Additionally, at this time, said subject will experience an enhanced antibody-mediated and T-cell immune response against NS3, as evidenced by the increased fraction of NS3-specific antibodies detected by EIA and a reduction in viral load detected by RT-PCR

Also contemplated are vaccine compositions comprising one or more of the HCV peptides described herein. Preferably, the encompassed peptide vaccines comprise ribavirin and NS3 / 4A, NS3

or a fragment or mutant thereof (SEQ ID NO: 2-26 and 36). The following example describes an approach by preparing a vaccine composition comprising an NS3 / 4A fusion protein and an adjuvant.

Example 10

By generating a labeled NS3 / 4A construct, the NS3 / 4A coding sequence of NS3 / 4A-pVAX or MSLF1-pVAX is removed enzymatically and the isolated fragment is inserted into an Xpress vector (Invitrogen). The Xpress vector allows the production of a recombinant fusion protein that has a short leader peptide in Nterminal that has a high affinity for divalent cations. Using a nickel chelating queen (Invitrogen), the recombinant protein can be purified in one step and the leader can then be removed by excision with enterokinase. A preferred vector is the pBlueBacHis2 Xpress. The pBlueBacHis2 Xpress vector is a Baculovirus expression vector that contains a multiple cloning site, an ampicillin resistance gene and a lac z gene. Accordingly, the digested amplification fragment is cloned into the pBlueBacHis2 Xpress vector and the SF59 cells are infected. Then, the expression protein is isolated or purified according to the manufacturer's instructions. Then, a vaccine composition containing NS3 / 4A is manufactured by mixing any amount of rNS3 / 4A between about 0.1-500 mg, for example 1-5 Ig, 5-10 Ig, 10-20 Ig, 20-30 Ig , 30-50 Ig, 50-100 Ig, 100-250 Ig or 250-500 Ig with any amount of ribavirin between approximately 0.1-10 mg, for example between 0.1 mg - 0.5 mg, 0.5 mg-1 mg, 1 mg-2 mg, 2 mg-5 mg or 5 mg-10 mg of ribavirin.

Said vaccine composition can be used to produce antibodies in a mammal (eg, mice or rabbits) or can be injected intramuscularly into a human being producing antibodies, preferably a human being who is chronically infected with the virus of the HCV The recipient preferably receives three immunization boosters of the mixture at 4 week intervals. Through the third booster, the specific HCV antibody titer will increase significantly. Additionally, at this time, said subject will experience an enhanced antibody-mediated and T-cell immune response against NS3, as evidenced by the increased fraction of NS3-specific antibodies detected by EIA and a reduction in viral load detected by RT-PCR

Compositions comprising one or more of the HCV nucleic acids or peptides covered may contain other ingredients, including, but not limited to, adjuvants, binding agents, excipients such as stabilizers (stimulating long-term storage), emulsifiers, thickeners, salts, preservatives, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delayed absorption agents and the like. These compositions are suitable for the treatment of animals, as a preventive measure avoiding a disease or condition or as a therapeutic product treating animals already affected by a disease or condition.

There may also be many other ingredients. For example, the adjuvant and the antigen can be used in admixture with conventional excipients (e.g., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application that do not react in a manner harmful with the adjuvant and / or the antigen). Suitable pharmaceutically acceptable carriers include, among others, water, saline solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silyl acid, paraffin viscose, perfume oil, monoglycerides and diglycerides of fatty acids, pentaerythritol ethers of fatty acids, hydroxymethyl cellulose, polyvinylpyrrolidone etc. Many more vehicles are described in Remmington's Pharmaceutical Sciences, 15th Edition, Easton: Mack Publishing Company, pages 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th Edition, Washington, American Pharmaceutical Association (1975).

The gene constructs described herein, in particular, can be formulated with or administered together with agents that increase uptake and / or expression of the gene construct by cells relative to the uptake and / or expression of the gene construct by the cells that are produced when the identical genetic vaccine is administered in the absence of such agents. Such agents and the protocols for administering them together with the gene constructs are described in the PCR patent application number PCT / US94 / 00899 filed on January 26, 1994. Examples of these agents include: CaPO4, DEAE dextran, anionic lipids, active enzymes. in the extracellular matrix, saponins, lectins, estrogenic compounds and steroid hormones, hydroxylated minor alkyls, dimethylsulfoxide (DMSO), urea and benzoic acid esters, anilides, amidines, urethanes and hydrochloride salts thereof, such as those of the family of local anesthetics. In addition, gene constructs are encapsulated or administered together with polycationic lipids / complexes.

The compositions described herein can be sterilized and, if desired, mixed with adjuvant agents, for example lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts influencing osmotic pressure, buffers, dyes, flavorings and / or substances aromatic and similar, that do not react detrimentally with the antigen or adjuvant.

The effective dose and method of administration of a specific formulation may vary based on each individual patient and the type and stage of the disease, as well as other factors known to those skilled in the art. The therapeutic efficacy and toxicity of vaccines can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example DE50 (the therapeutically effective dose in 50% of the population). Data obtained from cell culture assays and animal studies can be used in the formulation of a dosage range for human use. Vaccine doses are preferably within a range of circulating concentrations that include ED50 without toxicity. The dosage varies within this range depending on the type of adjuvant derivative and HCV antigen, the dosage form employed, the sensitivity of the patient and the route of administration.

Since many adjuvants, including ribavirin, have been on the market for several years, many dosage forms and routes of administration are known. All known dosage forms and routes of administration may be provided within the context of the embodiments described herein. Preferably, any amount of adjuvant that is effective in enhancing the immune response to an antigen in an animal can be considered to be any amount that is sufficient to reach a blood serum level of the antigen between 0.25 and 12.5 Ig / ml. in the animal, preferably about 2.5Ig / ml In some embodiments, the amount of adjuvant is determined according to the body weight of the animal to which the vaccine is to be administered. Accordingly, the amount of adjuvant in a particular formulation can be any amount between 0.1 - 6.0 mg / kg body weight. That is, some embodiments have an amount of adjuvant that corresponds to any amount between 0.1-1.0 mg / kg, 1.1-2.0 mg / kg, 2.1-3.0 mg / kg, 3 , 1 - 4.0 mg / kg, 4.1 - 5.0 mg / kg, 5.1 and 6.0 mg / kg of an animal's body weight. More conventionally, some of the compositions described herein contain any amount between 0.2 mg - 2000 mg of adjuvant. That is, some embodiments have approximately 250 Ig, 500 Ig, 1 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g , 1.6 g, 1.7 g, 1.8 g, 1.9 g and 2 g adjuvant.

As one skilled in the art will appreciate, the amount of antigens in a vaccine or immunogenic preparation may vary depending on the type of antigen and its immunogenicity. The amount of antigens in vaccines may vary accordingly. However, as a general guide, the compositions described herein may have any amount between about 0.25-2000 mg of an HCV antigen discussed herein. For example, the amount of antigen can be between about 0.25 nmg, 5-10 mg, 10-100 mg, 100-500 mg and higher than 2000 mg. Preferably, the amount of HCV antigen is 0.1 Ig-1 mg, desirably, 1 Ig-100 Ig, preferably 5 Ig-50 Ig and most preferably, 7 Ig, 8 Ig, 9 Ig, 10 Ig, 11 Ig20 Ig, when said antigen is a nucleic acid and 1 Ig-100 mg, desirably, 10 Ig-10 mg, preferably, 100 Ig-1 mg and most preferably, 200 Ig, 300 Ig, 400 Ig, 500 Ig, 600 Ig , or 700 Ig-1 mg, when said antigen is a peptide.

In some approaches described herein, the exact amount of adjuvant and / or HCV antigen is chosen by the individual physician according to the patient to be treated. In addition, the amounts of adjuvant can be added in combination with, or separately, of the same amount, or an equivalent, of antigen and these amounts can be adjusted during a particular vaccination protocol providing sufficient levels in light of specific considerations. of the patient or antigen specific. According to the above, patient-specific and antigen-specific factors that can be taken into account include, but are not limited to, the severity of the patient's illness, age and weight, diet, time and frequency of administration, the ( s) combinations of drugs, reaction sensitivities and tolerance / response to treatment. The following section describes the use of ribavirin as an adjuvant in greater detail.

Ribavirin

Nucleoside analogs have been widely used in antiviral therapies due to their ability to reduce viral replication. (Hosoya et al., J. Inf. Dis., 168: 641-646 (1993)). Ribavirin (1-1-D-ribofuranosyl-1,2,4-triazol-3carboxamide) is a synthetic analogue of guanosine that has been used to inhibit viral replication of RNA and DNA. (Huffman et al., Antimicrob. Agents. Chemother., 3: 235 (1973); Sidwell et al., Science, 177: 705 (1972)). Ribavirin has been shown to be a competitive inhibitor of inositol monophosphate (IMP) dehydrogenase (IMPDH), which converts IMP to IMX (which in turn becomes GMP). De Clercq, Anti viral Agents: characteristic activity spectrum depending on the molecular target with which they interact, Academic press, Inc., New York N.Y., p. 1-55 (1993). Intracellular groups of GTP are depleted as a result of prolonged treatment with ribavirin.

In addition to antiviral activity, researchers have noted that some guanosine analogues have an effect on the immune system. (U.S. Patent Nos. 6,063,772 and 4,950,647). Ribavirin has been shown to inhibit functional humoral immune responses (Peavy et al., J. Immunol., 126: 861-864 (1981); Powers et al., Antimicrob. Agents. Chemother., 22: 108-114 ( 1982)) and IgE-mediated modulation of mast cell secretion. (Marquardt et al., J. Pharmacol. Exp. Therapeutics, 240: 145-149 (1987)). Some researchers have reported that a daily oral treatment of ribavirin has an immune modulating effect on humans and mice. (Hultgren et al., J. Gen. Virol., 79: 2381-2391 (1998) and Cramp et al., Gastron. Enterol., 118: 346-355 (2000)). However, current knowledge of the effects of ribavirin on the immune system is in diapers. As disclosed below, ribavirin was found to be a potent adjuvant.

Example 11

In a first set of experiments, groups of three to five Balb / c mice (BK Universal, Uppsala, Sweden) i.p or s.c. were immunized. (e.g., at the base of the tail) with 10Ig or 100Ig of the recombinant non-structural protein 3 (rNS3) of the hepatitis C virus. The rNS3 was dissolved in phosphate buffered saline (PBS) alone or PBS containing 1 mg of ribavirin (obtained from ICN, Costa Mesa, CA). A total volume of 100 Il per injection was injected into the mice.

At two and four weeks after the i.p. immunization, blood was drawn from all mice by obtaining retroorbital samples. Serum samples were collected and analyzed for the presence of antibodies against rNS3. Determining the antibody titer an enzyme immunoassay (EIA) was performed. (See, for example, Hultgren et al., J Gen Virol. 79: 2381-91 (1998) and Hultgren et al., Clin. Diagn. Lab. Immunol.

5 4: 630-632 (1997)). Antibody levels were recorded as the highest serum dilution that gives an optical density at 405 nm more than twice that of the ratone are immunized.

Mice that received 10 Ig or 100 Ig of rNS3 mixed with 1 mg of ribavirin in PBS showed consistently higher levels of antibodies against NS3. The antibody titer that was detected by EIA a

10 the two weeks of immunization is shown in Figure 7. Vaccine formulations having 1 mg of ribavirin and 10 Ig or 100 Ig of rNS3 induced a significantly higher antibody titer than vaccine formulations composed solely of rNS3.

In a second set of experiments, groups of eight Balb / c mice were immunized intraperitoneally with

15 10 or 50 Ig of rNS3 in 100 Il of phosphate buffered saline solution containing 0 mg, 1 mg, 3 mg or 10 mg of ribavirin (Sigma). At four, six and eight weeks blood was drawn from the mice and the serum was separated and frozen. After completing the study, the sera were analyzed by determining the levels of antibodies against recombinant NS3, as described above. The average levels of antibodies against rNS3 were compared between the groups using Student's t-test (parametric analysis) or Mann-Whitney test (non-analysis

20 parametric) and the StatView 4.5 software package (Abacus Concepts, Berkely, CA). The adjuvant effect of ribavirin when added in three doses to 10 Ig of rNS3 is given in Table 11. The adjuvant effect of ribavirin when added in three doses to 50 Ig of rNS3 is given in Table 11. The comparison Parametric of the average titres of the antibodies against rNS3 in mice receiving different doses of 10 Ig or 50 Ig of and different doses of ribavirin is given in Tables 12 and 13, respectively. The nonparametric comparison

25 of the average titres of antibodies against NS3 in mice receiving different doses of 10 Ig or 50 Ig of rNS3 and different doses of ribavirin are given in Tables 14-16, respectively. The given values represent the final titers against recombinant rNS3.

TABLE 11 TABLE 12 TABLE 13

Amount of ribavirin (mg / dose)

Amount of immunogen (Ig / dose) Mouse ID Antibody titer against rNS3 the indicated week

Week 4

Week 6 Week 8

Any

10 5: 1 300 1500 1500

Any

10 5: 2 <60 7500 1500

Any

10 5: 3 <60 1500 300

Any

10 5: 4 60 1500 1500

Any

10 5: 5 <60 1500 nt

Any

10 5: 6 60 1500 1500

Any

10 5: 7 <60 7500 7500

Any

10 5: 8 300 37500 7500

Average group title (mean ± SD)

180 ± 139 7500 ± 12421 3042 ± 3076

one

10 6: 1 300 37500 37500

one

10 6: 2 <60 1500 1500

one

10 6: 3 300 37500 187500

one

10 6: 4 300 37500 7500

one

10 6: 5 60 nt nt

one

10 6: 6 <60 37500 7500

one

10 6: 7 <60 37500 7500

one

10 6: 8 300 7500 7500

Average group title (mean ± SD)

252 ± 107 28071 ± 16195 36642 ± 67565

3

10 7: 1 60 37500 7500

3

10 7: 2 60 37500 37500

3

10 7: 3 300 7500 7500

3

10 7: 4 300 37500 7500

3

10 7: 5 300 37500 37500

3

10 7: 6 300 37500 37500

3

10 7: 7 60 7500 7500

3 10 7: 8

60 37500 37500

Average group title (mean ± SD)

180 ± 128 30000 ± 13887 22500 ± 34637

10

10

8: 1 300 37500 37500

10

10

8: 2 300 37500 37500

10

10

8: 3 <60 300 300

10

10

8: 4 60 7500 7500

10

10

8: 5 <60 300 300

10

10

8: 6 <60 37500 37500

10

10

8: 7 <60 7500 7500

10

10

8: 8 <60 nt nt

Average group title (mean ± SD)

220 ± 139 18300 ± 18199 18300 ± 18199

Amount of ribavirin (mg / dose)

Amount of immunogen (Ig / dose) Mouse ID Antibody titer against rNS3 the indicated week

Week 4

Any

fifty 1: 1 60  Any fifty

Any

fifty 1: 2 60  Any fifty

Any

fifty 1: 3 60  Any fifty

Any

fifty 1: 4 <60  Any fifty

Any

fifty 1: 5 300  Any fifty

Any

fifty 1: 6 60  Any fifty

Any

fifty 1: 7 60  Any fifty

Any

fifty 1: 8  Any fifty

Average group title (mean ± SD)

100 ± 98 15214 ± 15380 10757 ± 12094

one

fifty 2: 1 60  one fifty

one

fifty 2: 2 300  one fifty

one

fifty 2: 3 60  one fifty

one

fifty 2: 4 60  one fifty

one

fifty 2: 5 60  one fifty

one

fifty 2: 6 60  one fifty

one

fifty 2: 7 300  one fifty

one

fifty 2: 8 300  one fifty

Average group title (mean ± SD)

150 ± 124 52500 ± 55549 37500 ± 62105

3

fifty 3: 1 60  3 fifty

3

fifty 3: 2 300  3 fifty

3

fifty 3: 3 300  3 fifty

3

fifty 3: 4 60  3 fifty

3

fifty 3: 5 300  3 fifty

3

fifty 3: 6 60  3 fifty

3

fifty 3: 7 -  3 fifty

3

fifty 3: 8 1500  3 fifty

Average group title (mean ± SD)

387 ± 513 30000 ± 13887 18750 ± 15526

10

fifty 4: 1 300  10 fifty

10

fifty 4: 2 300  10 fifty

10

fifty 4: 3 60  10 fifty

10

fifty 4: 4 60  10 fifty

10

fifty 4: 5 60  10 fifty

10

fifty 4: 6 60  10 fifty

10

fifty 4: 7 -  10 fifty

10

fifty 8: 8 60  10 fifty

Average group title (mean ± SD)

140 ± 124 10929 ± 11928 15214 ± 15380

Group

Week Mean ± SD  Group Mean ± SD Analysis P value

10 Ig of NS3 / without ribavirin

4 180 ± 139 10 Ig of NS3 / 1 mg of ribavirin 252 ± 107 Student t test from 0.4071

6

7500 ± 12421 28071 16195 ± Student t test from 0.0156 *

8

3042 ± 3076 36642 67565 ± Student t test from 0.2133

10 Ig of NS3 / without ribavirin

4 180 ± 139 10 Ig of NS3 / 3 mg of ribavirin 180 ± 128 Student t test from 1,000

6

7500 ± 12421 30000 13887 ± Student t test from 0.0042 **

8

3042 ± 3076 22500 34637 ± Student t test from 0.0077 **

10 Ig of NS3 / without ribavirin

4 180 ± 139 10 Ig of NS3 / 10mg of ribavirin 220 ± 139 Student t test from 0.7210

6

7500 ± 12421 18300 18199 ± Student t test from 0.1974

8

3042 ± 3076 18300 18199 ± Student t test from 0.0493 *

TABLE 14

Group

Week Mean ± SD  Group Mean ± SD Analysis P value

50 Ig of NS3 / without ribavirin

4 100 ± 98 50 Ig of NS3 / 1 mg of ribavirin 150 ± 1 24 Student t test from 0.4326

6

15214 ± 15380 52500 ± 55549 Student t test from 0,1106

8

10757 ± 12094 37500 ± 62105 Student t test from 0.2847

50 Ig of NS3 / without ribavirin

4 100 ± 98 50 Ig of NS3 / 3 mg of ribavirin 387 ± 513 Student t test from 0.2355

6

15214 ± 15380 30000 ± 13887 Student t test from 0.0721

8

10757 ± 12094 18750 ± 15526 Student t test from 0.2915

50 Ig of NS3 / without ribavirin

4 100 ± 98 50 Ig of NS3 / 10mg of ribavirin 140 ± 124 Student t test from 0.5490

6

15214 ± 15380 10929 ± 11928 Student t test from 0.5710

8

10757 ± 12094 15214 ± 15380 Student t test from 0.5579

Significant levels: NS = not significant; * = p <0.05; ** = p <0.01; *** = p <0.001

TABLE 15

Group

Week Mean ± SD  Group Mean ± SD Analysis P value

10 Ig of

4 180 ± 139  10 Ig of 252 ± 107 Mann 0.4280

NS3 / without

NS3 / 1 mg of Whitney

ribavirin

ribavirin

6

7500 ± 12421 28071 ± 16195 Mann-Whitney 0.0253 *

8

3042 ± 3076 36642 67565 ± Mann-Whitney 0.0245 *

10 Ig of

4 180 ± 139  10 Ig of 180 ± 128 Mann 0.0736

NS3 / without

NS3 / 3 mg of Whitney

ribavirin

ribavirin

6

7500 ± 12421 30000 13887 ± Mann-Whitney 0.0050 **

8

3042 ± 3076 22500 34637 ± Mann-Whitney 0.0034 **

10 Ig of

4 180 ± 139  10 Ig of 220 ± 139 Mann 0.8986

NS3 / without

NS3 / 10mg of Whitney

ribavirin

ribavirin

6

7500 ± 12421 18300 18199 ± Mann-Whitney 0.4346

8

3042 ± 3076 18300 18199 ± Mann-Whitney 0.2102

TABLE 16

Group

Week Mean ± SD  Group Mean ± SD Analysis P value

50 Ig of NS3 / without ribavirin

4 100 ± 98 50 Ig of NS3 / 1 mg of ribavirin 150 ± 124 Mann-Whitney 0,1128

6

15214 ± 15380 52500 ± 55549 Mann-Whitney 0.0210 *

8

10757 ± 12094 37500 ± 62105 Mann-Whitney 0.1883

50 Ig of NS3 / without ribavirin

4 100 ± 98 50 Ig of NS3 / 3 mg of ribavirin 387 ± 513 Mann-Whitney 0.1400

6

15214 ± 15380 30000 ± 13887 Mann-Whitney 0.0679

8

10757 ± 12094 18750 ± 15526 Mann-Whitney 0.2091

50 Ig of NS3 / without ribavirin

4 100 ± 98 50 Ig of NS3 / 10 mg of ribavirin 140 ± 124 Mann-Whitney 0.4292

6

15214 ± 15380 10929 ± 11928 Mann-Whitney 0.9473

8

10757 ± 12094 15214 ± 15380 Mann-Whitney 0.6279

Significant levels: NS = not significant; * = p <0.05; ** = p <0.01; *** = p <0.001

The previous data demonstrate that ribavirin facilitates or podenca an immune response to an antigen of the

5 HCV or HCV epitopes. Following immunization with a vaccine composition comprising only 1 mg of ribavirin and 10 Ig of the r rNS3 antigen, a potent immune response against rNS3 is produced. The above data also provide signs that the amount of ribavirin that is sufficient to facilitate an immune response to an antigen is between 1-3 mg per injection for a 25-30 g Balb / c mouse. However, it should be noted that these amounts are only as guides and should not be construed to limit the scope of the invention.

10 in any way. However, the data shows that vaccine compositions comprising approximately doses of 1 to 3 mg of ribavirin induce an immune response that is more than 112 times greater than the immune response produced in the absence of ribavirin. Therefore, ribavirin has a significant adjuvant effect on the humoral immune response of an animal and thus, enhances or facilitates the immune response to the antigen. The following example describes experiments that were performed to better understand

15 the amount of ribavirin needed by enhancing or facilitating an immune response to an antigen.

Example 12

Determining a dose of ribavirin that is sufficient to provide an adjuvant effect were performed.

20 following experiments. In a first set of experiments, groups of mice (three per group) were immunized with 20Ig of rNS3 alone or mixed as a mixture of 20Ig of rNS3 and 0.1 mg, 1 mg or 10 mg of ribavirin. Antibody levels against the antigen were determined by EIA. The mean final titers at weeks 1 and 3 were depicted and are shown in Figure 8. It was found that the adjuvant effect provided by ribavirin had different kinetics according to the dose of ribavirin provided. For example, even low doses (<1mg) are

25 found that they boosted antibody levels at week one, but not at week three, while higher doses (1-10 mg) were found to boost antibody levels at week three.

A second set of experiments was also performed. In these experiments, vaccine compositions comprising various amounts of ribavirin and rNS3 were injected into groups of mice and the

5 IgG response in these animals. The vaccine compositions comprised approximately 100 L of phosphate buffered saline and 20 Ig of rNS3 with or without 0.1 mg, 1.0 mg or 10 mg of ribavirin (Sigma). The blood was taken from the mice at week six and the specific IgG levels of rNS3 were determined by EIA as previously described. As shown in Table 17, the effects of the adjuvant on sustained levels and antibodies were more obvious in the dose range of 1 to 10 mg per injection for a 25-30 mg mouse.

10 TABLE 17

Immunogenic

Amount (mg) of ribavirin mixed with the immunogen Mouse ID Final IgG title of rNS3 the indicated week

Week 1

Week 2 Week 3

20 Ig of rNS3

Any one 60 360 360

20 Ig of rNS3

None 2 360 360 2160

20 Ig of rNS3

None 3 360 2160 2160

Half

260 ± 173 960 ± 1039 1560 ± 1039

20 Ig of rNS3

0.1 4 2160 12960 2160

20 Ig of rNS3

0.1 5 60 60 60

20 Ig of rNS3

0.1 6 <60 2160 2160

1110 ± 1484

5060 ± 6921 1460 ± 1212

20 Ig of rNS3

1.0 7 <60 60 12960

20 Ig of rNS3

1.0 8 <60 2160 2160

20 Ig of rNS3

1.0 9 360 2160 2160

Half

360 1460 ± 1212 5760 ± 6235

20 Ig of rNS3

10.0 10 360 12960 77760

20 Ig of rNS3

10.0 eleven <60 2160 12960

20 Ig of rNS3

10.0 12 360 2160 2160

Half

360 5760 ± 6235 30960 ± 40888

In a third set of experiments, the adjuvant effect of ribavirin after primary and booster injections was investigated. In these experiments, two intraperitoneal injections of a vaccine composition comprising 10 Ig of rNS3 with or without ribavirin were administered to the mice and IgG subclass responses to the antigen were monitored as before. Accordingly, mice were immunized with 100 Il phosphate buffer containing 10 Ig of recombinant NS3 alone, with or without 0.1 to 1.0 mg of ribavirin (Sigma) at weeks 0 and 4. Blood was drawn from the mice week six and the specific IgG subclasses of NS3 were determined by EIA as previously described. As shown in table 18, the addition of ribavirin to

The immunogen prior to injection does not modify the IgG subclass response in the specific immune response of NS3. Therefore, the adjuvant effect of a vaccine composition comprising ribavirin and an antigen cannot apply it as a change in the Th1 / Th2 imbalance. It seems that another mechanism may be responsible for the adjuvant effect of ribavirin.

TABLE 18 The data presented in this example also verify that ribavirin can be administered as an adjuvant and establish data that the dose of ribavirin can shape the kinetics of the effect without adjuvant. The following example describes another experiment that was performed to better understand the amount of ribavirin needed by potentiating or

Immunogenic

Amount (mg) of ribavirin mixed with the immunogen Mouse ID Final title of the subclass IgG of NS3 indicated

IgG1

IgG2a IgG2b IgG3

10 Ig of rNS3

Any one 360 60 <60 60

10 Ig of rNS3

Any 2 360 <60 <60 60

10 Ig of rNS3

Any 3 2160 60 <60 360

Half

960 ± 1039 60 - 160 ± 173

10 Ig of rNS3

0.1 4 360 <60 <60 60

10 Ig of rNS3

0.1 5 60 <60 <60 <60

10 Ig rNS3

0.1 6 2160 60 60 360

860 ± 1136

60 60 210 ± 212

10 Ig of rNS3

1.0 7 2160 <60 <60 60

10 Ig of rNS3

1.0 8 360 <60 <60 <60

10 Ig of rNS3

1.0 9 2160 <60 <60 60

Half

1560 ± 1039 - - 60

5 facilitating an immune response to an antigen.

Example 13

This assay can be used with any ribavirin derivative or combinations of ribavirin derivatives.

10 determining the extent that a particular vaccine formulation modulates an immune response. Determining the responses of CD4 + T cells to a vaccine containing ribavirin was immunized to groups of s.c. mice. with 100 Ig of rNS3 in PBS or 100 Ig of rNS3 and 1mg of ribavirin in PBS. Mice were sacrificed ten days after immunization and their lymph nodes were collected and drained, then in vitro memory tests were performed. (See, for example, Hultgren et al., J Gen Virol. 79: 2381-91 (1998) and Hultgren and

15 cols., Clin. Diag. Lab. Immunol. 4: 630-632 (1997)). The amount of proliferation of CD4 + T cells was determined at 96 h of culture by incorporation of thymidine [3H].

As shown in Figure 9, mice immunized with 100 Ig of rNS3 mixed with 1mg of ribavirin had a much larger T cell proliferative response than mice immunized with 100 Ig of rNS3 in

20 PBS These data provide more signs that ribavirin potentiates or facilitates a cellular immune response (eg, stimulating the effective stimulation of T cells).

Additional experiments were performed verifying that ribavirin enhances the immune response to commercially available vaccine preparations. The following example describes the use of ribavirin together with a preparation of

25 vaccines against commercial HBV.

Example 14 (Basic Example)

The adjuvant effect of ribavirin was analyzed when mixed with two available doses of a vaccine

30 commercially containing HBsAg and alumina. (Engerix, SKB). Approximately 0.2 Ig or 2 Ig of the Engerix vaccine was mixed with PBS or with 1 mg of ribavirin in PBS and the mixtures were injected intraperitoneally into groups of mice (three per group). A booster containing the same mixture was administered at week four and blood was drawn from all mice at the sixth week. Serum samples were diluted from 1:60 to 1: 37500 and the dilutions were analyzed by EIA, as described above, except that HBsAg was used

Human purified as the solid phase antigen. As shown in Table 19, vaccine formulations that have ribavirin boosted the 2 Ig response of an existing vaccine despite the fact that the vaccine already contained alumina. That is, by adding ribavirin to a suboptimal vaccine dose (i.e. one that does not induce detectable antibodies alone), the antibodies became detectable, which provided signs that the addition of ribavirin allows and use of amounts of antigen minors in a vaccine formulation that

40 compromise the immune response.

TABLE 19

Week

Final antibody titer against HBsAg in EIA

0.02 Ig of Engerix

0.2 Ig of Engerix

Ribavirin free

1 mg ribavirin  Ribavirin free 1 mg ribavirin

#one

#2 #3  #one #2 #3  #one #2 #3  #one #2 #3

6

<60 <60 <60 <60 <60 <60 <60 <60 <60 300 60 <60

The ribavirin used in the previous experiments was obtained from commercial suppliers (e.g., Sigma and ICN).

Ribavirin that can be used with the embodiments described herein can also be obtained from commercial suppliers or can be synthesized. Ribavirin and / or the antigen can be formulated with and without modification. For example, ribavirin may be modified or derivatized by making a more stable molecule and / or a more potent adjuvant. By one approach, the stability of ribavirin can be enhanced by coupling the molecules to a support such as a hydrophilic polymer (eg, polyethylene glycol).

Many more ribavirin derivatives can be generated using conventional techniques in rational drug design and combinatorial chemistry. For example, Molecular Simulations Inc. (MSI), as well as many other providers, provide software that allows the person skilled in the art to create a combinatorial library of organic molecules. For example, the C2.Analog Builder program can be integrated with an MSI suite of Cerius2 molecular diversity software by developing a library of ribavirin derivatives that can be used with the embodiments described herein. (See, for example, lhttp; // msi.com/life/products/cerius2/index.html).

Through an approach, the chemical structure of ribavirin is recorded in a computer-readable medium and accessed through one or more software application programs for modeling. The C2.Analog Builder program together with the C2Diversity program allows the user to generate a very large virtual library based on the diversity of the R groups for each substituent position. Compounds that have the same structure as the modeled ribavirin derivatives created in the virtual library are made using conventional chemistry or can be obtained from a commercial source.

The newly manufactured ribavirin derivatives can be screened in assays that determine the extent of the molecule's adjuvant activity and / or the extent of its ability by modulating an immune response. Some trials may involve virtual drug screening software, such as C2.Ludi. C2.Ludi is a software program that allows the user to explore databases of molecules (e.g., ribavirin derivatives) for their ability to interact with the active site of a protein of interest (e.g., .RAC2 or GTP binding molecules). Based on the interactions discovered with the virtual drug screening software, ribavirin derivatives may be a priority for subsequent characterization in conventional assays that determine the adjuvant activity and / or measurement of a molecule by modulating an immune response. The following section provides a major explanation. which concerns the procedures for using the compositions described herein.

Procedures for use of vaccine compositions and immunogenic preparations

The routes of administration of the embodiments described herein include, among others, the transdermal, parenteral, gastrointestinal, transbronchial and transalveolar pathways. Transdermal administration can be achieved by applying a rinse cream, gel etc. able to let the adjuvant and HCV antigen penetrate the skin. Parenteral routes of administration include, but are not limited to, electric or direct injection such as direct injection into a central venous, intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection. The gastrointestinal routes of administration include, among others, ingestion and rectal. Transbronchial and transalveolar administration routes include, among others, inhalation, either orally or intranasally.

Compositions having the HCV adjuvant and antigen that are suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams and ointments applied directly to the skin or incorporated into a protective vehicle such as a transdermal device ("patch" transdermal "). Examples of creams, ointments etc. Suitable can be found in, for example, the Physician's Desk Reference. Examples of suitable transdermal devices are described in, for example, US Pat. No. 4,818,540, filed April 4, 1989 by Moses et al.

Compositions having the HCV adjuvant and antigen that are suitable for parenteral administration include, among others, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, among others, phosphate buffered saline and oily preparations for injection in a central venous route, intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.

Compositions having the HCV adjuvant and antigen that are suitable for transbronchial and transalveolar administration include, among others, various types of aerosols for inhalation. Suitable devices for transbronchial and transalveolar administration of these are also embodiments. These devices include, among others, atomizers and vaporizers. Many forms of atomizers and vaporizers currently available can be easily adapted by releasing vaccines that have ribavirin and an antigen.

Compositions having the HCV adjuvant and antigen that are suitable for gastrointestinal administration include, among others, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration.

The gene constructs described herein, in particular, can be administered by means

including, among others, traditional syringes, needleless injection devices or "gene guns for microprojector bombing". Alternatively, the genetic vaccine can be introduced by various means into cells that are removed from the individual. Such means include, for example, ex vivo transfection, electroporation, microinjection or microprojector bombing. After the gene construct is captured by the cells, they are reimplanted in the individual. It is contemplated that cells, otherwise non-immunogenic, that have gene constructs incorporated therein can be implanted in the individual, even if the vaccinated cells initially came from another individual.

According to some embodiments, the gene construct is formulated by administering it to an individual using a needleless injection device. According to some embodiments, the gene construct is formulated for simultaneous administration to an individual intradermally, subcutaneously and intramuscularly using a needleless injection device. Needleless injection devices are well known and widely available. A person skilled in the art can, following the teachings herein, use needleless injection devices releasing the genetic material to the cells of an individual. Needleless injection devices are well adapted by releasing the genetic material to all tissues. They are particularly useful in releasing genetic material in the skin and muscle cells. A needleless injection device can be used to propel a liquid containing AD molecules to the surface of an individual's skin. The liquid is propelled at a sufficient speed in such a way that the impact with the skin, the liquid penetrates the surface of the skin, crosses the skin and muscle tissue between them. Therefore, the genetic material is administered simultaneously intradermally, subcutaneously or intramuscularly. A needleless injection device can be used releasing genetic material to the tissue of other organs in order to introduce a nucleic acid molecule to the cells to said organ.

Preferred embodiments refer to the use of a nucleic acid of the invention by preparing a medicament treating or preventing HCV infection. An animal that needs it will receive an HCV nucleic acid based antigen, as described herein (SEQ ID No. 35) and a sufficient amount of adjuvant exhibiting an adjuvant activity in said mammal. Accordingly, an animal can be identified as one that needs it using currently available diagnostic tests or clinical evaluation. The adjuvant and the antigen can be formulated for administration alone or in combinations and other adjuvants (eg, oil, alumina or other agents that enhance the immune response) can also be formulated for administration to an animal in need.

Preferred embodiments relate to the use of a nucleic acid of the invention by preparing a medicament enhancing an immune response against an HCV antigen in an animal that needs it, with an amount of adjuvant (e.g., ribavirin) and SEQ ID NO. 35 or a fragment thereof that is effective in enhancing said immune response. In these embodiments, an animal that needs a potentiated immune response against an antigen is identified using currently available diagnostic tests or clinical evaluation. By an approach, for example, the vaccine compositions described above are provided to an uninfected individual in a sufficient amount producing a cellular and humoral immune response to S3 so that said individual is protected from HCV infection. In another embodiment, an individual infected with HCV is identified by providing a vaccine composition comprising ribavirin and an NS3 in an amount sufficient to enhance the cellular and humoral immune response to NS3 so that HCV infection is reduced or eliminated. Said individual may be in the acute or chronic phase of the infection. In yet another embodiment, an HCV-infected individual suffering from HCC is provided with a composition comprising an adjuvant and the NS3 / 4A fusion gene in an amount sufficient to produce a cellular and humoral immune response against tumor cells expressing NS3. .

Sequence listing

<110> TRIPEP AB SALLBERG, Matti

<120> A FUSION GENE OF NS3 / 4A NON-STRUCTURAL OPTIMIZED BY CODON OF THE HEPATITIS C VIRUS

<130> TRIPEP.028QPC

<150> US 10 / 307,047

<151>

<160> 38

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 2061

<212> DNA

<213> Artificial sequence

<220>

<223> NS3 / 4A coding region of hepatitis C virus

<400> 1

<210> 2

<211> 686 10 <212> PRT

<213> Artificial sequence

<220>

<223> Hepatitis C virus NS3 / 4A peptide 15

<400> 2

<210> 3

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 3

<210> 4

<211> 686

<212> PRT

<213> Artificial sequence

5

<220>

<223> NS3 / 4A mutant hepatitis C virus

<400> 4

10

<210> 5

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 5

<210> 6

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 6 <210> 7

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 7

<210> 8

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 8

<210> 9

<211> 686 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant hepatitis C virus

<400> 9

<210> 10

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 10

<210> 11

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A mutant of hepatitis C virus 10

<400> 11 <210> 12

<211> 632 5 <212> PRT

<213> Artificial sequence

<220>

<223> Hepatitis C virus NS3 peptide 10

<400> 12

<210> 13

<211> 54 5 <212> PRT

<213> Artificial sequence

<220>

<223> Hepatitis C virus NS4A peptide 10

<400> 13

15 <210> 14

<211> 25

<212> PRT

<213> Artificial sequence

20 <220>

<223> Hepatitis C virus NS3 / 4A peptide

<400> 14

<210> 15

<211> 12

<212> PRT 30 <213> Artificial sequence

<220>

<223> Hepatitis C virus NS3 / 4A peptide

35 <400> 15 <210> 16

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 16

<210> 17

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 17

<210> 18

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 18

<210> 19

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 19

<210> 20

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 20

<210> 21

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 21

<210> 22

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 22

<210> 23

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 23

<210> 24

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 24 <210> 25

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS3 / 4A Peptide

<400> 25

<210> 26

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Hepatitis C Virus NS5A / B Peptide

<400> 26

<210> 27

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Hepatitis C virus NS5 peptide

<400> 27

<210> 28

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 28

ccgtctagat cagcactctt ccatttcatc 30

<210> 29

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 29

cctgaattca tggcgcctat cacggcctat 30

<210> 30

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 30

ccacgcggcc gcgacgacct acag 24

<210> 31

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 31

ctggaggtcg tcacgcctac ctgggtgctc gtt 33

<210> 32

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 32

accgagcacc caggtaggcg tgacgacctc cag 33

<210> 33

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 33

ctggaggtcg tccgcggtac ctgggtgctc gtt 33

<210> 34

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Cloning oligonucleotide

<400> 34

accgagcacc caggtaccgc ggacgacctc cag 33

<210> 35

<211> 2078

<212> DNA <213> Artificial sequence

<220>

<223> Codon optimized codon-optimized hepatitis C virus NS3 / 4A coding region

<221> CDS

<222> (12) ... (2072)

<400> 35

<210> 36

<211> 686 5 <212> PRT

<213> Artificial sequence

<220>

<223> NS3 / 4A coding region of hepatitis C virus optimized by codon 10

<400> 36

<210> 37

<211> 9

<212> PRT

<213> Artificial sequence <220>

<223> Artificial peptide, NS3 / 4A H-2D binding peptide

<400> 37

<210> 38

<211> 9 10 <212> PRT

<213> Artificial sequence

<220>

<223> Artificial peptide, H-2D control peptide 15

<400> 38

Claims (20)

one.

 A purified or isolated nucleic acid comprising at least 200 consecutive nucleotides of the NS3 / 4A gene of SEQ ID No. 35 or its complement, wherein said nucleic acid is capable of inducing an immune response against HCV NS3 / 4A.

2.

 A purified or isolated nucleic acid comprising at least 50 consecutive nucleotides of the NS3 / 4A gene of SEQ ID No. 35 or its complement, wherein said nucleic acid is capable of inducing an immune response against HCV NS3 / 4A and with the condition that said nucleic acid does not have the sequence set forth in SEQ ID NO: 3, 10 or 11 disclosed in WO 03/031588 or the sequence set forth in SEQ ID NO: 21 disclosed in WO 2004/046175.

3.

 The purified or isolated nucleic acid of claim 2, wherein said nucleic acid comprises at least 60 consecutive nucleotides of the NS3 / 4A gene of SEQ ID No. 35 or its complement.

Four.

 The purified or isolated nucleic acid of claim 2, wherein said nucleic acid comprises at least 100 consecutive nucleotides of the NS3 / 4A gene of SEQ ID No. 35 or its complement.

5.

 The purified or isolated nucleic acid of claim 1, wherein said nucleic acid comprises at least 500 consecutive nucleotides of the NS3 / 4A gene of SEQ ID NO: 35 or its complement.

6.

 The purified or isolated nucleic acid of claim 1, wherein said nucleic acid comprises an NS3 / 4A gene of SEQ ID No. 35 or its complement.

7.

 The purified or isolated nucleic acid of claim 1, wherein said nucleic acid consists of the NS3 / 4A gene of SEQ ID No. 35 or its complement.

8.

 A vector comprising the purified or isolated nucleic acid according to any one of claims 1-7.

9.

An immunogenic composition comprising the nucleic acid of any one of claims 1-7.

10.

 The immunogenic composition of claim 9, further comprising an adjuvant.

eleven.

 The immunogenic composition of claim 9, further comprising ribavirin.

12.

 An isolated cell comprising the nucleic acid of any one of claims 1-7.

13.

 The nucleic acid of any one of claims 1-7, the vector of claim 8, the immunogenic composition of any one of claims 9-11 or the isolated cell of claim 12 for use as a medicament.

14.

 The use of the nucleic acid of any one of claims 1-7, the vector of claim 8, the isolated cell of claim 12, or the immunogenic composition of claims 9-11 to prepare a medicament for the purpose of generating a immune response to NS3 / 4A of HCV.

fifteen.

 The use of the nucleic acid of any one of claims 1-7, the vector of claim 8, the isolated cell of claim 12, or the immunogenic composition of claims 9-11 to prepare a medicament for the purpose of treatment or HCV infection prevention.

16.

 The use of the nucleic acid of any one of claims 1-7, the vector of claim 8, or the immunogenic composition of claims 9-10 for the preparation of a medicament for the treatment

or prevention of HCV infection, in which said medication is to be administered alone or together with a ribavirin, which can be administered separately rather than in a single mixture.

17.

 The use according to any one of claims 15 or 16, wherein said medicament is for the treatment of hepatocellular carcinoma (CHC) or a chronic or acute phase HCV infection.

18.

The process for manufacturing an immunogen comprising:

provide ribavirin providing a nucleic acid according to any one of claims 1-7, or the vector of claim 8; combining said ribavirin and said nucleic acid or said vector so that said immunogen is formulated.

19.

 The composition according to any one of claims 1-11 or the use of claims 14 or 15, wherein said nucleic acid is formulated for administration by transdermal, intradermal, intravenous, subcutaneous, intramuscular, intraperitoneal or inhalation route.

twenty.

 The composition according to any one of claims 1-11 or the use of claims 14 or 15, wherein said nucleic acid is formulated for administration by electroporation, microparticle bombardment or needleless injection.